Antiproliferative compounds, conjugates thereof, methods therefor, and uses thereof

ABSTRACT

Antiproliferative compounds having a structure represented by formula (II), where n, R 1 , R 2 , R 3 , R 4 , and R 5  are as defined herein, can be used to treat tumors, optionally when conjugated to a ligand such as an antibody:

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of USProvisional Applications Nos. 61/230,932, filed Aug. 3, 2009; and61/232,883, filed Aug. 11, 2009; the disclosure of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention relates to compounds structurally related to thetubulysins, conjugates thereof with a ligand, methods for making andusing such compounds and conjugates, and compositions comprising suchcompounds and conjugates.

The tubulysins are cytotoxins originally isolated from cultures of themyxobacteria Archangium gephyra or Angiococcus disciformis, with eachorganism producing a different mixture of tubulysins (Sasse et al. 2000;Reichenbach et al. 1998). Their crystal structure and biosyntheticpathway have been elucidated (Steinmetz et al. 2004) and theirbiosynthesis genes have been sequenced (Hoefle et al. 2006b).Pretubulysin, a biosynthetic precursor of the tubulysins, also has beenshown to possess significant activity in its own right (Ullrich et al.2009). (Full citations for the documents cited herein by first author orinventor and year are listed at the end of this specification.)

The tubulysins belong to a group of naturally occurring antimitoticpolypeptides and depsipeptides that includes the phomopsins, thedolastatins, and the cryptophycins (Hamel 2002). Antimitotic agentsother than polypeptides or depsipeptides also exist, for examplepaclitaxel, the maytansines, and the epothilones. During mitosis, acell's microtubules reorganize to form the mitotic spindle, a processrequiring the rapid assembly and disassembly of the microtubuleconstituent proteins α- and β-tubulin. Antimitotic agents block thisprocess and prevent a cell from undergoing mitosis, although at themolecular level the exact blockage mechanism may differ from one agentto another. The tubulysins prevent the assembly of the tubulins intomicrotubules, causing the affected cells to accumulate in the G₂/M phaseand undergo apoptosis (Khalil et al. 2006). Conversely, paclitaxeleffects the same end result by binding to microtubules and preventingtheir disassembly.

The tubulysins have a tetrapeptidyl scaffold constructed from oneproteinogenic and three non-proteinogenic amino acid subunits:N-methylpipecolinic acid (Mep), isoleucine (Ile), tubuvaline (Tuv), andeither tubuphenylalanine (Tup, R^(A) equals H in formula (I) below) ortubutyrosine (Tut, R^(A) equals OH). About a dozen naturally occurringtubulysins (named A, B, etc.) are known, the sites of structuralvariation among them being at residues R^(A), R^(B) and R^(C) as shownin Formula (I) and Table 1:

TABLE 1 Naturally Occurring Tubulysins Tubulysin R^(A) R^(B) R^(C) A OHOC(═O)Me CH₂OC(═O)i-Bu B OH OC(═O)Me CH₂OC(═O)n-Pr C OH OC(═O)MeCH₂OC(═O)Et D H OC(═O)Me CH₂OC(═O)i-Bu E H OC(═O)Me CH₂OC(═O)n-Pr F HOC(═O)Me CH₂OC(═O)Et G OH OC(═O)Me CH₂OC(═O)CH═CH₂ H H OC(═O)MeCH₂OC(═O)Me I OH OC(═O)Me CH₂OC(═O)Me U H OC(═O)Me H V H OH H Z OH OH HPretubulysin H H Me

Kaur et al. 2006 studied the antiproliferative properties of tubulysin Aand found that it was more potent than other antimitotic agents such aspaclitaxel and vinblastine and was active in xenograft assays against avariety of cancer cell lines. Further, tubulysin A induced apoptosis incancer cells but not normal cells and showed significant potentialantiangiogenic properties in in vitro assays. The antimitotic propertiesof other tubulysins have also been evaluated and generally have beenfound to compare favorably against those of non-tubulysin antimitoticagents (see, e.g., Balasubramanian et al. 2009; Steinmetz et al. 2004;Wipf et al. 2004). For these reasons, there is considerable interest inthe tubulysins as anti-cancer agents (see, e.g., Domling et al. 2005c;Hamel 2002).

Numerous publications describe efforts directed at the synthesis oftubulysins, including: Balasubramanian et al. 2009; Domling et al. 2006;Hoefle et al. 2003; Neri et al. 2006; Peltier et al. 2006; Sani et al.2007; Sasse et al. 2007; Shankar et al. 2009; Shibue et al. 2009; andWipf et al. 2004. Other publications describe structure-activityrelationship (SAR) studies, via the preparation and evaluation oftubulysin analogs or derivatives: Balasubramanian et al. 2008 and 2009;Domling 2006; Domling et al. 2005a; Ellman et al. 2009; Hoefle et al.2001 & 2006a; Patterson et al. 2007 & 2008; Richter 2008; Vlahov et al.2009; Wang et al. 2007; and Wipf et al. 2007 and 2010. The SAR studiesmainly explored structural variations in the Mep ring, residues R^(B)and R^(C) of the Tuv subunit, and the aromatic ring or aliphatic carbonchain of the Tup/Tut subunit.

Domling et al. 2005 disclose conjugates of tubulysins with a partnermolecule generically described as a polymer or a biomolecule, but withactual examples limited to polyethylene glycol (PEG) as the partnermolecule. Other documents disclosing conjugates of tubulysins are Boydet al. 2008 and 2010; Vlahov et al. 2008a, 2008b and 2010; Leamon et al.2008 and 2009; Reddy et al. 2009; and Low et al. 2009. Leung et al. 2002disclose polyanionic polypeptides that can be conjugated to drugs(including tubulysins) to improve their bioactivity and watersolubility.

Davis et al. 2008 and Schluep et al. 2009 disclose cyclodextrin basedformulations in which tubulysins are covalently attached to acyclodextrin via a hydrazide-disulfide linker moiety bonded to theTup/Tut carboxyl group.

BRIEF SUMMARY OF THE INVENTION

The present invention discloses novel antiproliferative compounds thatare structurally related to the tubulysins, are cytotoxic or cytostaticagainst many cancer cells, and are believed to act by an antimitoticmechanism. These compounds can be conjugated to ligands such asantibodies for targeted delivery against cancer cells.

In one embodiment, this invention provides a compound having a structurerepresented by formula (II)

wherein

-   -   n is 0, 1, or 2;    -   R¹, R² and R³ are independently H, unsubstituted or substituted        C₁-C₁₀ alkyl, unsubstituted or substituted C₂-C₁₀ alkenyl,        unsubstituted or substituted C₂-C₁₀ alkynyl, unsubstituted or        substituted aryl, unsubstituted or substituted heteroaryl,        unsubstituted or substituted (CH₂)₁₋₂O(C₁-C₁₀ alkyl),        unsubstituted or substituted (CH₂)₁₋₂O(C₂-C₁₀ alkenyl),        unsubstituted or substituted (CH₂)₁₋₂O(C₂-C₁₀ alkynyl),        (CH₂)₁₋₂OC(═O)(C₁-C₁₀ alkyl), unsubstituted or substituted        (CH₂)₁₋₂OC(═O)(C₂-C₁₀ alkenyl), unsubstituted or substituted        (CH₂)₁₋₂OC(═O)(C₂-C₁₀ alkynyl), unsubstituted or substituted        C(═O)(C₁-C₁₀ alkyl), unsubstituted or substituted C(═O)(C₂-C₁₀        alkenyl), unsubstituted or substituted C(═O)(C₂-C₁₀ alkynyl),        unsubstituted or substituted cycloaliphatic, unsubstituted or        substituted heterocycloaliphatic, unsubstituted or substituted        arylalkyl, or unsubstituted or substituted alkylaryl;    -   R⁴ is

and

-   -   R⁵ is H, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, CO(C₁-C₅        alkyl), CO(C₂-C₅ alkenyl), or CO(C₂-C₅ alkynyl);        or a pharmaceutically acceptable ester thereof, a        pharmaceutically acceptable amide thereof at the carboxyl group        of R⁴ with the α-amino group of an α-amino acid, or a        pharmaceutically acceptable salt thereof.

A preferred R⁴ is

with the stereochemistry at the methyl group alpha to the carboxyl beingmore preferably that corresponding to the natural tubulysins, that is:

This invention also provides novel intermediates useful for synthesizingcompounds according to formula (II).

In another embodiment, this invention provides a compound of thisinvention conjugated via a linker moiety to a ligand (preferably anantibody, more preferably a monoclonal antibody, and most preferably ahuman monoclonal antibody) for its selective delivery to a target cellsuch as a cancer cell.

In another embodiment, there is provided a composition of mattercomprising a compound of this invention and a linker moiety, suitablefor conjugation to a ligand.

In another embodiment, this invention provides a method for inhibitingthe proliferation of cancer cells in a subject suffering from cancer,comprising administering to the subject a therapeutically effectiveamount of a compound of this invention or a conjugate thereof with aligand (particularly an antibody). In another embodiment, there isprovided a method for inhibiting the proliferation of cancer cells,comprising contacting such cells with a compound of this invention or aconjugate thereof with a ligand (particularly an antibody), underconditions sufficient to inhibit the growth of such cancer cells. Thecancer cells can be colorectal cancer, liver cancer, prostate cancer,breast cancer, melanoma, glioblastoma, lung cancer, pancreatic cancer,ovarian cancer, multiple myeloma, renal cancer, leukemia, or lymphomacells. Where the ligand is an antibody, it is preferred that theantibody binds to an antigen expressed by the cancer cells.

In another embodiment, there is provided a method of treating a cancerin a subject suffering from such cancer, comprising administering to thesubject a therapeutically effective amount of a compound of thisinvention or a conjugate thereof with a ligand (particularly anantibody). In another embodiment, there is provided the use of acompound of this invention (or a conjugate thereof with a ligand(particularly an antibody) for the preparation of a medicament for thetreatment of cancer. In these embodiments, the cancer can be colorectalcancer, liver cancer, prostate cancer, breast cancer, melanoma,glioblastoma, lung cancer, pancreatic cancer, ovarian cancer, multiplemyeloma, renal cancer, leukemia, or lymphoma. Where the ligand is anantibody, it is preferred that the antibody binds to an antigenexpressed by the cells of the cancer.

In another embodiment, there is provided the use of a compound of thisinvention or a conjugate thereof with a ligand (preferably an antibody)for the preparation of a medicament for treating a cancer in a subjectsuffering from such cancer.

BRIEF DESCRIPTION OF THE DRAWING(S)

FIGS. 1 a and 1 b depict, in combination, Scheme 1 for making compoundsof this invention.

FIGS. 2 and 3 depict Schemes 2 and 3, respectively, also for makingcompounds of this invention.

FIG. 4 depicts a Scheme 4 suitable for attaching a peptidyl linker and amaleimide reactive group to compounds of this invention.

FIGS. 5, 6, and 7 depict Schemes 5, 6, and 7, respectively, for makingcompounds of this invention.

FIGS. 8 a, 8 b, and 8 c show Schemes 8, 9, and 10, respectively, formaking intermediates useful for preparing compounds of this invention.

FIGS. 9 and 10 show Schemes 11 and 12, respectively, illustrating howintermediates such as those shown in FIGS. 8 a-8 c can be elaboratedinto compounds of this invention.

FIGS. 11 a and 11 b show the plots for ³H thymidine proliferation assaysfor a first set of compounds of this invention, against two differenttypes of cancer cells.

FIGS. 12 a and 12 b show the plots for ATP luminescence proliferationassays for a second set of compounds of this invention, against twodifferent types of cancer cells. FIGS. 12 c and 12 d show the plots for³H thymidine proliferation assays for the same second set of compoundsand against the same two types of cancer cells.

FIG. 13 shows the activity against renal cancer cells of conjugates ofcompounds of this invention in ³H thymidine proliferation assays.

FIG. 14 shows the activity against renal cancer cells of conjugates ofcompounds of this invention in xenograft studies.

FIG. 15 shows a Scheme 13 for making intermediates useful for makingcompounds of this invention.

FIG. 16 shows a Scheme 14 for making compounds of this invention fromintermediates made per Scheme 13.

FIGS. 17 and 18 show Schemes 15 and 16, respectively, for makingconjugation-ready compounds of this invention.

FIG. 19 shows Scheme 17 for making an intermediate useful for makingcompounds of this invention.

FIGS. 20 a and 20 b show in combination Scheme 18 for the preparation ofcompounds of this invention from the intermediate of Scheme 17.

FIG. 21 shows Scheme 19 for making an intermediate used in Scheme 18.

FIG. 22 shows Scheme 20 for the synthesis of an intermediate used inmaking compounds of this invention.

FIG. 23 shows Scheme 21 for making compounds of this invention from theintermediate of Scheme 20.

FIG. 24 shows the Scheme 22 for making yet another intermediate usefulfor making compounds of this invention.

FIG. 25 shows Scheme 23 for making compounds of this invention from theintermediate of Scheme 22.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Antibody” means whole antibodies and any antigen binding fragment(i.e., “antigen-binding portion”) or single chains thereof. A wholeantibody is a glycoprotein comprising at least two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds. Each heavychain comprises a heavy chain variable region (V_(H)) and a heavy chainconstant region comprising three domains, C_(H1), C_(H2) and C_(H3).Each light chain comprises a light chain variable region (V_(L) orV_(k)) and a light chain constant region comprising one single domain,C_(L). The V_(H) and V_(L) regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDRs), interspersed with more conserved framework regions (FRs). EachV_(H) and V_(L) comprises three CDRs and four FRs, arranged from amino-to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. The variable regions contain a binding domain thatinteracts with an antigen. The constant regions may mediate the bindingof the antibody to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system. An antibody is said to “specificallybind” to an antigen X if the antibody binds to antigen X with a K_(D) of5×10⁻⁸ M or less, more preferably 1×10⁻⁸ M or less, more preferably6×10⁻⁹ M or less, more preferably 3×10⁻⁹ M or less, even more preferably2×10⁻⁹ M or less. The antibody can be chimeric, humanized, or,preferably, human. The heavy chain constant region can be engineered toaffect glycosylation type or extent, to extend antibody half-life, toenhance or reduce interactions with effector cells or the complementsystem, or to modulate some other property. The engineering can beaccomplished by replacement, addition, or deletion of one or more aminoacids or by replacement of a domain with a domain from anotherimmunoglobulin type, or a combination of the foregoing.

“Antibody fragment” and “antigen-binding portion” of an antibody (orsimply “antibody portion”) mean one or more fragments of an antibodythat retain the ability to specifically bind to an antigen. It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody, such as (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H1)domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fab′fragment, which is essentially an Fab with part of the hinge region(see, for example, Abbas et al., Cellular and Molecular Immunology, 6thEd., Saunders Elsevier 2007); (iv) a Fd fragment consisting of the V_(H)and C_(H1) domains; (v) a Fv fragment consisting of the V_(L) and V_(H)domains of a single arm of an antibody, (vi) a dAb fragment (Ward etal., (1989) Nature 341:544-546), which consists of a V_(H)domain; (vii)an isolated complementtarity determining region (CDR); and (viii) ananobody, a heavy chain variable region containing a single variabledomain and two constant domains. Furthermore, although the two domainsof the Fv fragment, V_(L) and V_(H), are encoded by separate genes, theycan be joined, using recombinant methods, by a synthetic linker thatenables them to be made as a single protein chain in which the V_(L) andV_(H) regions pair to form monovalent molecules (known as single chainFv, or scFv); see, e.g., Bird et al. (1988) Science 242:423-426; andHuston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Suchsingle chain antibodies are also encompassed within the term“antigen-binding portion” of an antibody.

An “isolated antibody” means an antibody that is substantially free ofother antibodies having different antigenic specificities (e.g., anisolated antibody that specifically binds antigen X is substantiallyfree of antibodies that specifically bind antigens other than antigenX). An isolated antibody that specifically binds antigen X may, however,have cross-reactivity to other antigens, such as antigen X moleculesfrom other species. In certain embodiments, an isolated antibodyspecifically binds to human antigen X and does not cross-react withother (non-human) antigen X antigens. Moreover, an isolated antibody maybe substantially free of other cellular material and/or chemicals.

“Monoclonal antibody” or “monoclonal antibody composition” means apreparation of antibody molecules of single molecular composition, whichdisplays a single binding specificity and affinity for a particularepitope.

“Human antibody” means an antibody having variable regions in which boththe framework and CDR regions (and the constant region, if present) arederived from human germline immunoglobulin sequences. Human antibodiesmay include later modifications, including natural or syntheticmodifications. Human antibodies may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, “human antibody” does not include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

“Human monoclonal antibody” means an antibody displaying a singlebinding specificity, which has variable regions in which both theframework and CDR regions are derived from human germline immunoglobulinsequences. In one embodiment, human monoclonal antibodies are producedby a hybridoma that includes a B cell obtained from a transgenicnonhuman animal, e.g., a transgenic mouse, having a genome comprising ahuman heavy chain transgene and a light chain transgene fused to animmortalized cell.

“Aliphatic” means a straight- or branched-chain, saturated orunsaturated, non-aromatic hydrocarbon moiety having the specified numberof carbon atoms (e.g., as in “C₃ aliphatic,” “C₁-C₅ aliphatic,” or “C₁to C₅ aliphatic,” the latter two phrases being synonymous for analiphatic moiety having from 1 to 5 carbon atoms) or, where the numberof carbon atoms is not explicitly specified, from 1 to 4 carbon atoms (2to 4 carbons in the instance of unsaturated aliphatic moieties).

“Alkyl” means a saturated aliphatic moiety, with the same convention fordesignating the number of carbon atoms being applicable. By way ofillustration, C₁-C₄ alkyl moieties include, but are not limited to,methyl, ethyl, propyl, isopropyl, isobutyl, t-butyl, 1-butyl, 2-butyl,and the like.

“Alkenyl” means an aliphatic moiety having at least one carbon-carbondouble bond, with the same convention for designating the number ofcarbon atoms being applicable. By way of illustration, C₂-C₄ alkenylmoieties include, but are not limited to, ethenyl (vinyl), 2-propenyl(allyl or prop-2-enyl), cis-1-propenyl, trans-1-propenyl, E- (or Z-)2-butenyl, 3-butenyl, 1,3-butadienyl (but-1,3-dienyl) and the like.

“Alkynyl” means an aliphatic moiety having at least one carbon-carbontriple bond, with the same convention for designating the number ofcarbon atoms being applicable. By way of illustration, C₂-C₄ alkynylgroups include ethynyl (acetylenyl), propargyl (prop-2-ynyl),1-propynyl, but-2-ynyl, and the like.

“Cycloaliphatic” means a saturated or unsaturated, non-aromatichydrocarbon moiety having from 1 to 3 rings, each ring having from 3 to8 (preferably from 3 to 6) carbon atoms. “Cycloalkyl” means acycloaliphatic moiety in which each ring is saturated. “Cycloalkenyl”means a cycloaliphatic moiety in which at least one ring has at leastone carbon-carbon double bond. “Cycloalkynyl” means a cycloaliphaticmoiety in which at least one ring has at least one carbon-carbon triplebond. By way of illustration, cycloaliphatic moieties include, but arenot limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, and adamantyl.Preferred cycloaliphatic moieties are cycloalkyl ones, especiallycyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

“Heterocycloaliphatic” means a cycloaliphatic moiety wherein, in atleast one ring thereof, up to three (preferably 1 to 2) carbons havebeen replaced with a heteroatom independently selected from N, O, or S,where the N and S optionally may be oxidized and the N optionally may bequaternized. Similarly, “heterocycloalkyl,” “heterocycloalkenyl,” and“heterocycloalkynyl” means a cycloalkyl, cycloalkenyl, or cycloalkynylmoiety, respectively, in which at least one ring thereof has been somodified. Exemplary heterocycloaliphatic moieties include aziridinyl,azetidinyl, 1,3-dioxanyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl,piperidinyl, piperazinyl, tetrahydropyranyl, tetrahydrothiopyranyl,tetrahydrothiopyranyl sulfone, morpholinyl, thiomorpholinyl,thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxolanyl,tetrahydro-1,1-dioxothienyl, 1,4-dioxanyl, thietanyl, and the like.

“Alkoxy,” “aryloxy,” “alkylthio,” and “arylthio” mean —O(alkyl),—O(aryl), —S(alkyl), and —S(aryl), respectively. Examples are methoxy,phenoxy, methylthio, and phenylthio, respectively.

“Halogen” or “halo” means fluorine, chlorine, bromine or iodine.

“Aryl” means a hydrocarbon moiety having a mono-, bi-, or tricyclic ringsystem wherein each ring has from 3 to 7 carbon atoms and at least onering is aromatic. The rings in the ring system may be fused to eachother (as in naphthyl) or bonded to each other (as in biphenyl) and maybe fused or bonded to non-aromatic rings (as in indanyl orcyclohexylphenyl). By way of further illustration, aryl moietiesinclude, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl,indanyl, biphenyl, phenanthryl, anthracenyl, and acenaphthyl.

“Heteroaryl” means a moiety having a mono-, bi-, or tricyclic ringsystem wherein each ring has from 3 to 7 carbon atoms and at least onering is an aromatic ring containing from 1 to 4 heteroatomsindependently selected from N, O, or S, where the N and S optionally maybe oxidized and the N optionally may be quaternized. Such at least oneheteroatom containing aromatic ring may be fused to other types of rings(as in benzofuranyl or tetrahydroisoquinolyl) or directly bonded toother types of rings (as in phenylpyridyl or 2-cyclopentylpyridyl). Byway of further illustration, heteroaryl moieties include pyrrolyl,furanyl, thiophenyl (thienyl), imidazolyl, pyrazolyl, oxazolyl,isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, pyridyl,N-oxopyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, quinolinyl,isoquinolynyl, quinazolinyl, cinnolinyl, quinozalinyl, naphthyridinyl,benzofuranyl, indolyl, benzothiophenyl, oxadiazolyl, thiadiazolyl,phenothiazolyl, benzimidazolyl, benzotriazolyl, dibenzofuranyl,carbazolyl, dibenzothiophenyl, acridinyl, and the like.

Where it is indicated that a moiety may be substituted, such as by useof “substituted or unsubstituted” or “optionally substituted” phrasingas in “substituted or unsubstituted C₁-C₅ alkyl” or “optionallysubstituted heteroaryl,” such moiety may have one or more independentlyselected substituents, preferably one to five in number, more preferablyone or two in number. Substituents and substitution patterns can beselected by one of ordinary skill in the art, having regard for themoiety to which the substituent is attached, to provide compounds thatare chemically stable and that can be synthesized by techniques known inthe art as well as the methods set forth herein.

“Arylalkyl,” (heterocycloaliphatic)alkyl,” “arylalkenyl,” “arylalkynyl,”“biarylalkyl,” and the like mean an alkyl, alkenyl, or alkynyl moiety,as the case may be, substituted with an aryl, heterocycloaliphatic,biaryl, etc., moiety, as the case may be, with the open (unsatisfied)valence at the alkyl, alkenyl, or alkynyl moiety, for example as inbenzyl, phenethyl, N-imidazoylethyl, N-morpholinoethyl, and the like.Conversely, “alkylaryl,” “alkenylcycloalkyl,” and the like mean an aryl,cycloalkyl, etc., moiety, as the case may be, substituted with an alkyl,alkenyl, etc., moiety, as the case may be, for example as inmethylphenyl (tolyl) or allylcyclohexyl. “Hydroxyalkyl,” “haloalkyl,”“alkylaryl,” “cyanoaryl,” and the like mean an alkyl, aryl, etc.,moiety, as the case may be, substituted with one or more of theidentified substituent (hydroxyl, halo, etc., as the case may be).

By way of illustration, permissible substituents include, but are notlimited to, alkyl (especially methyl or ethyl), alkenyl (especiallyallyl), alkynyl, aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic,halo (especially fluoro), haloalkyl (especially trifluoromethyl),hydroxyl, hydroxyalkyl (especially hydroxyethyl), cyano, nitro, alkoxy,—O(hydroxyalkyl), —O(haloalkyl) (especially —OCF₃), —O(cycloalkyl),—O(heterocycloalkyl), —O(aryl), alkylthio, arylthio, ═O, ═NH, ═N(alkyl),═NOH, ═NO(alkyl), —C(═O)(alkyl), —C(═O)H, —CO₂H, —C(═O)NHOH,—C(═O)O(alkyl), —C(═O)O(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl),—C(═O)N(alkyl)₂, —OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)O(alkyl),—OC(═O)O(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,azido, —NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NH(hydroxyalkyl),—NHC(═O)(alkyl), —NHC(═O)H, —NHC(═O)NH₂, —NHC(═O)NH(alkyl),—NHC(═O)N(alkyl)₂, —NHC(═NH)NH₂, —OSO₂(alkyl), —SH, —S(alkyl), —S(aryl),—S(cycloalkyl), —S(═O)alkyl, —SO₂(alkyl), —SO₂NH₂, —SO₂NH(alkyl),—SO₂N(alkyl)₂, and the like.

Where the moiety being substituted is an aliphatic moiety, preferredsubstituents are aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic,halo, hydroxyl, cyano, nitro, alkoxy, —O(hydroxyalkyl), —O(haloalkyl),—O(cycloalkyl), —O(heterocycloalkyl), —O(aryl), alkylthio, arylthio, ═O,═NH, ═N(alkyl), ═NOH, ═NO(alkyl), —CO₂H, —C(═O)NHOH, —C(═O)O(alkyl),—C(═O)O(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl), —C(═O)N(alkyl)₂,—OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)O(alkyl),—OC(═O)O(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,azido, —NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NH(hydroxyalkyl),—NHC(═O)(alkyl), —NHC(═O)H, —NHC(═O)NH₂, —NHC(═O)NH(alkyl),—NHC(═O)N(alkyl)₂, —NHC(═NH)NH₂, —OSO₂(alkyl), —SH, —S(alkyl), —S(aryl),—S(═O)alkyl, —S(cycloalkyl), —SO₂(alkyl), —SO₂NH₂, —SO₂NH(alkyl), and—SO₂N(alkyl)₂. More preferred substituents are halo, hydroxyl, cyano,nitro, alkoxy, —O(aryl), ═O, ═NOH, ═NO(alkyl), —OC(═O)(alkyl),—OC(═O)O(alkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂, azido,—NH₂, —NH(alkyl), —NH(alkyl)₂, —NH(aryl), —NHC(═O)(alkyl), —NHC(═O)H,—NHC(═O)NH₂, —NHC(═O)NH(alkyl), —NHC(═O)N(alkyl)₂, and —NHC(═NH)NH₂.

Where the moiety being substituted is a cycloaliphatic,heterocycloaliphatic, aryl, or heteroaryl moiety, preferred substituentsare alkyl, alkenyl, alkynyl, halo, haloalkyl, hydroxyl, hydroxyalkyl,cyano, nitro, alkoxy, —O(hydroxyalkyl), —O(haloalkyl), —O(aryl),—O(cycloalkyl), —O(heterocycloalkyl), alkylthio, arylthio,—C(═O)(alkyl), —C(═O)H, —CO₂H, —C(═O)NHOH, —C(═O)O(alkyl),—C(═O)O(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl), —C(═O)N(alkyl)₂,—OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)O(alkyl),—OC(═O)O(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,azido, —NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NH(hydroxyalkyl),—NHC(═O)(alkyl), —NHC(═O)H, —NHC(═O)NH₂, —NHC(═O)NH(alkyl),—NHC(═O)N(alkyl)₂, —NHC(═NH)NH₂, —OSO₂(alkyl), —SH, —S(alkyl), —S(aryl),—S(cycloalkyl), —S(═O)alkyl, —SO₂(alkyl), —SO₂NH₂, —SO₂NH(alkyl), and—SO₂N(alkyl)₂. More preferred substituents are alkyl, alkenyl, halo,haloalkyl, hydroxyl, hydroxyalkyl, cyano, nitro, alkoxy,—O(hydroxyalkyl), —C(═O)(alkyl), —C(═O)H, —CO₂H, —C(═O)NHOH,—C(═O)O(alkyl), —C(═O)O(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl),—C(═O)N(alkyl)₂, —OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)O(alkyl),—OC(═O)O(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,—NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NHC(═O)(alkyl), —NHC(═O)H,—NHC(═O)NH₂, —NHC(═O)NH(alkyl), —NHC(═O)N(alkyl)₂, and —NHC(═NH)NH₂.

Where a range is stated, as in “C₁-C₅ alkyl” or “5 to 10%,” such rangeincludes the end points of the range, as in C₁ and C₅ in the firstinstance and 5% and 10% in the second instance.

Unless particular stereoisomers are specifically indicated (e.g., by abolded or dashed bond at a relevant stereocenter in a structuralformula, by depiction of a double bond as having E or Z configuration ina structural formula, or by use stereochemistry-designatingnomenclature), all stereoisomers are included within the scope of theinvention, as pure compounds as well as mixtures thereof. Unlessotherwise indicated, individual enantiomers, diastereomers, geometricalisomers, and combinations and mixtures thereof are all encompassed bythe present invention.

Those skilled in the art will appreciate that compounds may havetautomeric forms (e.g., keto and enol forms), resonance forms, andzwitterionic forms that are equivalent to those depicted in thestructural formulae used herein and that the structural formulaeencompass such tautomeric, resonance, or zwitterionic forms.

“Pharmaceutically acceptable ester” means an ester that hydrolyzes invivo (for example in the human body) to produce the parent compound or asalt thereof or has per se activity similar to that of the parentcompound. Suitable esters include C₁-C₅ alkyl, C₂-C₅ alkenyl or C₂-C₅alkynyl esters, especially methyl, ethyl or n-propyl.

“Pharmaceutically acceptable salt” means a salt of a compound suitablefor pharmaceutical formulation. Where a compound has one or more basicgroups, the salt can be an acid addition salt, such as a sulfate,hydrobromide, tartrate, mesylate, maleate, citrate, phosphate, acetate,pamoate (embonate), hydroiodide, nitrate, hydrochloride, lactate,methylsulfate, fumarate, benzoate, succinate, mesylate, lactobionate,suberate, tosylate, and the like. Where a compound has one or moreacidic groups, the salt can be a salt such as a calcium salt, potassiumsalt, magnesium salt, meglumine salt, ammonium salt, zinc salt,piperazine salt, tromethamine salt, lithium salt, choline salt,diethylamine salt, 4-phenylcyclohexylamine salt, benzathine salt, sodiumsalt, tetramethylammonium salt, and the like. Polymorphic crystallineforms and solvates are also encompassed within the scope of thisinvention.

Compounds

A preferred embodiment of compounds of formula (II) is represented byformula (II-a)

wherein n, R¹, R², and R³ are as defined hereinabove in respect offormula (II) and R^(4a) is

In the compounds of formula (II-a), the subunit corresponding to Tup/Tutin the naturally occurring tubulysins has been reduced in size andlipophilicity by at least two carbons, via deletion of the two aliphaticcarbon atoms immediately following the carboxylic acid group—that is,the amino group is now α- to the carboxylic acid group, instead of γ- toit. In the instance in which R⁴ is 4-aminophenylalanine, the amine groupconstitutes a polar moiety that further reduces lipophilicity. SARstudies show that lipophilicity is an important factor in the biologicalactivity of the tubulysins and their analogs or derivatives. Steinmetzet al. 2004 and Neri et al. 2006 both disclose that the more lipophilicnaturally occurring tubulysins—i.e., those having a Tup subunit (R^(A)equals H in formula I) instead of a Tut subunit (R^(A) equals OH informula I)—possessed greater biological activity. Further, thedifferences in activities were retained regardless of the size andlipophilicity of the 11-acyloxy residue (group R^(C) in formula (I)) inthe Tuv subunit (Steinmetz et al. 2004). These results indicate that alipophilic Tup/Tut subunit is a particularly important SAR element.

The above observations are partially corroborated in two studies byBalasubramanian et al. In the first (Balasubramanian et al. 2008),analogs dimethylated at the carbon alpha to the carboxyl group in theTup subunit were compared against otherwise identical analogsdesmethylated at the same position. The dimethylated analogs had greaterantiproliferative activity—although the in vitro tubulin inhibitionIC₅₀s were comparable—as might be expected based on their relativelipophilicities. However, this trend was not followed in the secondstudy (Balasubramanian et al. 2009), in which three analogs (oneα-carbon desmethylated, one monomethylated, and one dimethylated) werecompared. There, the most active analog was the desmethylated one whilethe monomethylated one—i.e., with the natural Tup subunit—was by far theleast active one. However, the latter analog had additionalmodifications elsewhere in the molecule rendering it essentiallyinactive, making it unclear what SAR inferences, if any, can be drawn.

Patterson et al. 2007 and Ellman et al. 2009 compared the cytotoxicitiesof tubulysin D and analogs in which either only the phenethyl or theγ-carboxy group was retained in the Tup subunit. The phenethyl-retainedanalog was 3.6 to 13.6 times less active than tubulysin D against threecancer cell lines but when the less lipophilic γ-carboxyl group wasretained, there was an even greater loss of activity, from 25.7 to 62.5times less active. That is, the order of activity was:

The above documents suggest, individually and in combination, thatlipophilicity at the Tup locus is especially important for thebiological activity of the tubulysins. Therefore, the prior art suggeststhat the replacement of the Tup subunit with a phenylalanine (Phe),4-aminophenylalanine (4-NH₂Phe), norvaline or other R⁴ subunit inaccordance with formula (II-a) is undesirable, as each would lead to theloss of at least two aliphatic carbons and a consequent reduction inlipophilicity at the Tup/Tut locus.

Another preferred embodiment of compounds of formula (II) is representedby formula (II-b):

wherein n, R¹, R², and R³ are as defined hereinabove in respect forformula (II). Although chemical formulae can be found in the literatureencompassing an —NH₂ group in the 4-position of the Tup aromatic ring(Domling 2005a and 2005b), there has been no disclosure on how acompound having such a feature might be made.

In formulae (II), (II-a), and (II-b), R¹ preferably is H, C₁-C₅ alkyl,C₂-C₅ alkenyl, or C₂-C₅ alkenyl, and is more preferably an isoleucylresidue, that is:

In formulae (II), (II-a), and (II-b), R² preferably is H, C₁-C₅ alkyl,C₂-C₅ alkenyl, CH₂O(C₁-C₅ alkyl), CH₂O(C₂-C₅ alkenyl), CH₂O(C═O)(C₁-C₅alkyl), or CH₂OC(═O)(C₂-C₅ alkenyl); and more preferably is H, Me, Et,n-Pr, CH₂OMe, CH₂OEt, CH₂O(n-Pr), CH₂OC(═O)i-Bu, CH₂OC(═O)n-Pr,CH₂OC(═O)CH═CH₂, or CH₂OC(═O)Me, with Me, n-Pr, CH₂OMe, CH₂OC(═O)i-Bu,and CH₂O(n-Pr) being especially preferred.

In formulae (II), (II-a), and (II-b), R³ preferably is H, C₁-C₅ alkyl,C₂-C₅ alkenyl, C(═O)C₁-C₅ alkyl, or C(═O)C₂-C₅ alkenyl; and morepreferably is H, Me, Et, or C(═O)Me.

Preferably, in formulae (II) and (II-a), R⁴ and R^(4a) are:

In formulae (II), (II-a),and (II-b), n preferably is 1 and, in theinstance of formula (II), R⁵ preferably is methyl; that is, the ring inthe Mep subunit preferably is an N-methyl piperidinyl one.

A preferred embodiment of compounds according to formula (II-a) isdepicted by formula (II-a′)

where R^(4a) is as defined above in respect of formula (II-a), R² is H,C₁-C₅ alkyl, C₂-C₅ alkenyl, CH₂O(C₁-C₅ alkyl), CH₂O(C₂-C₅ alkenyl),CH₂O(C═O)(C₁-C₅ alkyl), or CH₂OC(═O)(C₂-C₅ alkenyl); and R³ is H, C₁-C₅alkyl, C₂-C₅ alkenyl, C(═O)C₁-C₅ alkyl, or C(═O)C₂-C₅ alkenyl.Preferably, R² is H, Me, Et, n-Pr, CH₂OMe, CH₂OEt, CH₂OC(═O)i-Bu,CH₂OC(═O)n-Pr, CH₂OC(═O)CH═CH₂, or CH₂OC(═O)Me; more preferably Me,n-Pr, CH₂OMe, CH₂OC(═O)i-Bu, or CH₂O(n-Pr). Preferably, R³ is H, Me, Et,or C(═O)Me.

A preferred embodiment of compounds according to formula (II-b) isdepicted by formula (II-b′)

where R² is H, C₁-C₅ alkyl, C₂-C₅ alkenyl, CH₂O(C₁-C₅ alkyl), CH₂O(C₂-C₅alkenyl), CH₂O(C═O)(C₁-C₅ alkyl), or CH₂OC(═O)(C₂-C₅ alkenyl); and R³ isH, C₁-C₅ alkyl, C₂-C₅ alkenyl, C(═O)C₁-C₅ alkyl, or C(═O)C₂-C₅ alkenyl.Preferably, R² is H, Me, Et, n-Pr, CH₂OMe, CH₂OEt, CH₂OC(═O)i-Bu,CH₂OC(═O)n-Pr, CH₂OC(═O)CH═CH₂, or CH₂OC(═O)Me and R³ is H, Me, Et, orC(═O)Me.

Where a carboxyl group in R⁴ is esterified, preferably the ester is aC₁-C₅ alkyl ester, such as a Me, Et, or Pr ester. Alternatively, thecarboxyl group can be amidated with ammonia or an alkyl amine

In another embodiment, this invention provides a compound having astructure represented by formula (II-c)

where R¹³ is Me, n-Pr, CH₂OMe, or CH₂OC(═O)CH₂CH(Me)₂; R¹⁴ is Me orC(═O)Me; and R¹⁵ is H or C₁-C₅ alkyl (preferably H, Me, or Et).

In formulae (II-b), (II-b′), and (II-c) the stereochemistry at themethyl alpha to the carboxyl preferably is that corresponding to thenaturally-occurring tubulysins, i.e.:

In a preferred embodiment, a compound of this invention is in the formof an amide of the carboxyl group in R⁴ (or R^(4a), as the case may be)with the α-amine group of an α-amino acid. The α-amino acid can beselected from the group consisting of a proteinogenic amino acid,4-aminophenylalanine, norvaline, norleucine, and citrulline. Preferably,the α-amino acid is selected from the group consisting of alanine,norvaline, glycine, lysine, arginine, citrulline, norleucine,4-aminophenylalanine, and phenylalanine. Also preferably, the absoluteconfiguration of the α-amino acid is the proteinogenic one, i.e., L. Inthis preferred embodiment, R⁴ (or R^(4a)) preferably is:

Specific examples of compounds of this invention according to formula(II) include compounds (III-a) through (III-y). Some of the compoundsare depicted as a pharmaceutically acceptable ester or apharmaceutically acceptable amide of the R⁴ carboxyl group with theα-amine group of an α-amino acid or methyl ester thereof

This invention also provides novel intermediates that can be utilizedfor the synthesis of compounds of this invention. Compounds according toformula (VIII-a) can be used for the making of compounds according toformula (II) or (II-b), as taught in the figures and examples herein.

In formula (VIII-a), R⁷ is H or an amine protecting group and R⁸ is H,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, aryl, cycloaliphatic,alkylcycloaliphatic, arylalkyl, or alkylaryl. Preferably, R⁷ is H, Boc(t-butoxycarbonyl), Troc (2,2,2-trichloroethoxy carbonyl), Bpoc((1-methyl-1-(4-biphenyl)ethoxycarbonyl)), Cbz (benzyloxy carbonyl),Aloc (allyloxycarbonyl), methyl amine, or Fmoc(9-fluorenylmethoxycarbonyl). Preferably, R⁸ is H or C₁-C₅ alkyl(especially Me).

Another novel intermediate useful for synthesis of compounds of thisinventtion has a structure according to formula (VIII-b) The use ofcompounds of formula (VIII-b) to make compounds of this invention istaught in the figures and examples herein.

In formula (VIII-b), R⁹ and R¹⁰ are independently H or an amineprotecting group and R¹¹ is H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, aryl, cycloaliphatic, alkylcycloaliphatic, arylalkyl, oralkylaryl. Preferably, R⁹ and R¹⁰ are independently selected from H,Boc, Troc, Bpoc, Cbz, Aloc, methylamine, and Fmoc. Preferably, R¹¹ is Hor C₁-C₅ alkyl (especially Me). Preferably, where R⁹ and R¹⁰ are each anamine protecting group, they are different amine protecting groups.

Additional suitable amine protecting groups for the compounds of formula(VIII-a) and (VIII-b) are disclosed in Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3rd edition, pp. 464-653 (John Wiley &Sons, New York, 1999), the disclosure of which is incorporated herein byreference.

Conjugates

In another aspect, there is provided a conjugate comprising cytotoxiccompound according to this invention and a ligand, represented byformula (IV)

[D(X^(D))_(a)C(X^(Z))_(b)]_(m)Z  (IV)

where Z is ligand; D is a cytotoxic compound according to thisinvention; and —(X^(D))_(a)C(X^(Z))_(b)— are collectively referred to asa “linker moiety” or “linker” because they link Z and D. Within thelinker, C is a cleavable group designed to be cleaved at the site ofintended biological action of compound D; X^(D) and X^(Z) are referredto as spacer moieties (or “spacers”) because they space apart D and Cand C and Z, respectively; subscripts a and b are independently 0 or 1(that is, the presence of X^(D) and/or X^(Z) are optional); andsubscript m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (preferably 1, 2, 3, or4). D, X^(D), C, X^(Z) and Z are more fully defined hereinbelow.

Ligand Z—for example an antibody—serves a targeting function. By bindingto a target tissue or cell where its antigen or receptor is located,ligand Z directs the conjugate there. Preferably, the target tissue orcell is a cancer tissue or cell and the antigen or receptor is atumor-associated antigen, that is, an antigen that is uniquely expressedby cancerous cells or is overexpressed by cancer cells, compared tonon-cancerous cells. Cleavage of group C at the target tissue or cellreleases compound D to exert its cytotoxic effect locally. In someinstances, the conjugate is internalized into a target cell byendocytosis and cleavage takes place within the target cell. In thismanner, precise delivery of compound D is achieved at the site ofintended action, reducing the dosage needed. Also, compound D isnormally biologically inactive (or significantly less active) in itsconjugated state, thereby reducing undesired toxicity against non-targettissue or cells. As anticancer drugs are often highly toxic to cells ingeneral, this is an important consideration.

As reflected by the subscript m, each molecule of ligand Z can conjugatewith more than one compound D, depending on the number of sites D hasavailable for conjugation and the experimental conditions employed.Those skilled in the art will appreciate that, while each individualmolecule of ligand Z is conjugated to an integer number of compounds D,a preparation of the conjugate may analyze for a non-integer ratio ofcompounds D to ligand Z, reflecting a statistical average.

Ligand Z and Conjugation Thereof

Preferably, ligand Z is an antibody. For convenience and brevity and notof limitation, the detailed subsequent discussion herein about theconjugation of ligand Z is written in the context of its being anantibody, but those skilled in the art will understand that other typesof ligand Z can be conjugated, mutatis mutandis. For example, conjugateswith folic acid as the ligand can target cells having the folatereceptor on their surfaces (Vlahov et al. 2008a, 2008b and 2010; Leamonet al. 2009). For the same reason, the detailed discussion below isprimarily written in terms of a 1:1 ratio of antibody Z to compound D.

Preferably, ligand Z is an antibody against a tumor associated antigen,allowing a conjugate comprising such a ligand Z to selectively targetcancer cells. Examples of such antigens include: mesothelin, prostatespecific membrane antigen (PSMA), CD19, CD22, CD30, CD70, B7H4 (alsoknown as O8E), protein tyrosine kinase 7 (PTK7), RG1, CTLA-4, and CD44.The antibody can be animal (e.g., murine), chimeric, humanized, or,preferably, human. The antibody preferably is monoclonal, especially amonoclonal human antibody. The preparation of human monoclonalantibodies against some of the aforementioned antigens is disclosed inKorman et al., US 2009/0074660 A1 (B7H4); Rao-Naik et al., US2009/0142349 A1 A2 (CD19); King et al., WO 2008/070569 A2 (CD22); Keleret al., U.S. Pat. No. 7,387,776 B2 (2008) (CD30); Terrett et al., US2009/0028872 A1 (CD70); Korman et al., U.S. Pat. No. 6,984,720 B1 (2006)(CTLA-4); Korman et al., US 2009/0217401 A1 (PD-1); Huang et al., US2008/0279868 A1 (PSMA); Lu et al., US 2010/0034826 A1 (PTK7); Harkins etal., U.S. Pat. No. 7,335,748 B2(2008) (RG1); Terrett et al., WO2009/045957 A1 (mesothelin); and Xu et al., US 2010/0092484 A1 (CD44);the disclosures of which are incorporated herein by reference.

Ligand Z can also be an antibody fragment or antibody mimetic, such asan affibody, a domain antibody (dAb), a nanobody, a unibody, a DARPin,an anticalin, a versabody, a duocalin, a lipocalin, or an avimer.

Any one of several different reactive groups on ligand Z can be aconjugation site, including ε-amino groups in lysine residues, pendantcarbohydrate moieties, carboxylic acid groups, disulfide groups, andthiol groups. Each type of reactive group represents a trade-off, havingsome advantages and some disadvantages. For reviews on antibody reactivegroups suitable for conjugation, see, e.g., Garnett, Adv. Drug DeliveryRev. 53 (2001), 171-216 and Dubowchik and Walker, Pharmacology &Therapeutics 83 (1999), 67-123, the disclosures of which areincorporated herein by reference.

In one embodiment, ligand Z is conjugated via a lysine ε-amino group.Most antibodies have multiple exposed lysine ε-amino groups, which canbe conjugated via amide, urea, thiourea, or carbamate bonds usingtechniques known in the art, including modification with aheterobifunctional agent (as further described hereinbelow). However, itis difficult to control which and how many ε-amino groups react, leadingto potential batch-to-batch variability in conjugate preparations. Also,conjugation may cause neutralization of a protonated ε-amino groupimportant for maintaining the antibody's native conformation or may takeplace at a lysine near or at the antigen binding site, neither being adesirable occurrence.

In another embodiment, ligand Z can be conjugated via a carbohydrateside chain, as many antibodies are glycosylated. The carbohydrate sidechain can be oxidized with periodate to generate aldehyde groups, whichin turn can be reacted with amines to form an imine group, such as in asemicarbazone, oxime, or hydrazone. If desired, the imine group can beconverted to a more stable amine group by reduction with sodiumcyanoborohydride. For additional disclosures on conjugation viacarbohydrate side chains, see, e.g., Rodwell et al., Proc. Nat'l Acad.Sci. USA 83, 2632-2636 (1986); the disclosure of which is incorporatedherein by reference. As with lysine ε-amino groups, there are concernsregarding reproducibility of the location of the conjugation site(s) andstoichiometry.

In yet another embodiment, ligand Z can be conjugated via a carboxylicacid group. In one embodiment, a terminal carboxylic acid group isfunctionalized to generate a carbohydrazide, which is then reacted withan aldehyde-bearing conjugation moiety. See Fisch et al., BioconjugateChemistry 1992, 3, 147-153.

In yet another embodiment, antibody Z can be conjugated via a disulfidegroup bridging a cysteine residue on antibody Z and a sulfur on theother portion of the conjugate. Some antibodies lack free thiol(sulfhydryl) groups but have disulfide groups, for example in the hingeregion. In such case, free thiol groups can be generated by reduction ofnative disulfide groups. The thiol groups so generated can then be usedfor conjugation. See, e.g., Packard et al., Biochemistry 1986, 25,3548-3552; King et al., Cancer Res. 54, 6176-6185 (1994); and Doroninaet al., Nature Biotechnol. 21(7), 778-784 (2003); the disclosures ofwhich are incorporated herein by reference. Again, there are concernsregarding conjugation site location and stoichiometry and the possibledisruption of antibody native conformation.

A number of methods are known for introducing free thiol groups intoantibodies without breaking native disulfide bonds, which methods can bepracticed with a ligand Z of this invention. Depending on the methodemployed, it may be possible to introduce a predictable number of freesulfhydryls at predetermined locations. In one approach, mutatedantibodies are prepared in which a cysteine is substituted for anotheramino acid. See, for example, Eigenbrot et al., US 2007/0092940 A1;Chilkoti et al., Bioconjugate Chem. 1994, 5, 504-507; Urnovitz et al.,U.S. Pat. No. 4,698,420 (1987); Stimmel et al., J. Biol. Chem., 275(39), 30445-30450 (2000); Bam et al., U.S. Pat. No. 7,311,902 B2 (2007);Kuan et al., J. Biol. Chem., 269 (10), 7610-7618 (1994); Poon et al., J.Biol. Chem., 270 (15), 8571-8577 (1995). In another approach, an extracysteine is added to the C-terminus See, e.g. Cumber et al., J.Immunol., 149, 120-126 (1992); King et al, Cancer Res., 54, 6176-6185(1994); Li et al., Bioconjugate Chem., 13, 985-995 (2002); Yang et al.,Protein Engineering, 16, 761-770 (2003); and Olafson et al., ProteinEngineering Design & Selection, 17, 21-27 (2004). A preferred method forintroducing free cysteines is that taught by Liu et al., WO 2009/026274A1, in which a cysteine bearing amino acid sequence is added to theC-terminus of the heavy chain of an antibody. This method introduces aknown number of cysteine residues (one per heavy chain) at a knownlocation remote from the antigen binding site. The disclosures of thedocuments cited in this paragraph are all incorporated herein byreference.

In yet another embodiment, lysine ε-amino groups can be modified withheterobifunctional reagents such as 2-iminothiolane orN-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP), converting anε-amino group into a thiol or disulfide group—creating a cysteinesurrogate, as it were. However, this method suffers from the sameconjugation location and stoichiometry limitations associated withε-amino groups proper.

In yet another preferred embodiment, ligand Z is conjugated via thenucleophilic addition product of a thiol group to an acceptor moiety. Apreferred acceptor moiety is a maleimide group, whose reaction with anantibody thiol group is generically illustrated below. The thiol groupcan be a native one, or one introduced as described above.

Linker—(X^(D))_(a)C(X^(Z))_(b)—

As noted above, the linker portion of a conjugate of this inventioncomprises up to three elements: a cleavable group C and optional spacersX^(Z) and X^(D).

Cleavable group C is a group cleavable under physiological conditions,preferably is selected such that it is relatively stable while theconjugate is in general circulation in the blood plasma, but is readilycleaved once the conjugate reaches its site of intended action, that is,near, at, or within the target cell. Preferably, the conjugate isinternalized by endocytosis by a target cell upon binding of antibody Zto an antigen displayed on the surface of the target cell. Subsequently,cleavage of group C occurs in a vesicular body of the target cell (anearly endosome, a late endosome, or, especially, a lysosome).

In one embodiment, group C is a pH sensitive group. The pH in bloodplasma is slightly above neutral, while the pH inside a lysosome isacidic, circa 5. Thus, a group C whose cleavage is acid catalyzed willcleave at a rate several orders of magnitude faster inside a lysosomethan in the blood plasma rate. Examples of suitable acid-sensitivegroups include cis-aconityl amides and hydrazones, as described in Shenet al., U.S. Pat. No. 4,631,190 (1986); Shen et al., U.S. Pat. No.5,144,011 (1992); Shen et al., Biochem. Biophys. Res. Commun. 102,1048-1054 (1981) and Yang et al., Proc. Natl Acad. Sci (USA), 85,1189-1193 (1988); the disclosures of which are incorporated herein byreference.

In another embodiment, group C is a disulfide. Disulfides can be cleavedby a thiol-disulfide exchange mechanism, at a rate dependent on theambient thiol concentration. As the intracellular concentration ofglutathione and other thiols is higher than their serum concentrations,the cleavage rate of a disulfide will be higher intracellularly.Further, the rate of thiol-disulfide exchange can be modulated byadjustment of the steric and electronic characteristics of the disulfide(e.g., an alkyl-aryl disulfide versus an alkyl-alkyl disulfide;substitution on the aryl ring, etc.), enabling the design of disulfidelinkages that have enhanced serum stability or a particular cleavagerate. For additional disclosures relating to disulfide cleavable groupsin conjugates, see, e.g., Thorpe et al., Cancer Res. 48, 6396-6403(1988); Santi et al., US 2005/0287155 A1; Ng et al., U.S. Pat. No.6,989,452 B2 (2006); Ng et al., WO 2002/096910 A1; Boyd et al., U.S.Pat. No. 7,691,962 B2; and Sufi et al., WO 2008/083312 A2; thedisclosures of which are incorporated herein by reference.

A preferred group C comprises a peptide bond that is cleaved,preferentially by a protease, at the intended site of action, as opposedto by a protease in the serum. Typically, group C comprises from 1 to 20amino acids, preferably from 1 to 6 amino acids, more preferably from 1to 3 amino acids. The amino acid(s) can be natural and/or unnaturalα-amino acids. Natural amino acids are those encoded by the geneticcode, as well as amino acids derived therefrom, e.g., hydroxyproline,γ-carboxyglutamate, citrulline, and O-phosphoserine. The term amino acidalso includes amino acid analogs and mimetics. Analogs are compoundshaving the same general H₂N(R)CHCO₂H structure of a natural amino acid,except that the R group is not one found among the natural amino acids.Examples of analogs include homoserine, norleucine,methionine-sulfoxide, and methionine methyl sulfonium. An amino acidmimetic is a compound that has a structure different from the generalchemical structure of an α-amino acid but functions in a manner similarto one. The term “unnatural amino acid” is intended to represent the “D”stereochemical form, the natural amino acids being of the “L” form.

Preferably, group C contains an amino acid sequence that is a cleavagerecognition sequence for a protease. Many cleavage recognition sequencesare known in the art. See, e.g., Matayoshi et al. Science 247: 954(1990); Dunn et al. Meth. Enzymol. 241: 254 (1994); Seidah et al. Meth.Enzymol. 244: 175 (1994); Thornberry, Meth. Enzymol. 244: 615 (1994);Weber et al. Meth. Enzymol. 244: 595 (1994); Smith et al. Meth. Enzymol.244: 412 (1994); and Bouvier et al. Meth. Enzymol. 248: 614 (1995); thedisclosures of which are incorporated herein by reference.

For conjugates that are not intended to be internalized by a cell, agroup C can be chosen such that it is cleaved by a protease present inthe extracellular matrix in the vicinity of the target tissue, e.g., aprotease released by nearby dying cells or a tumor-associated protease.Exemplary extracellular tumor-associated proteases are matrixmetalloproteases (MMP), thimet oligopeptidase (TOP) and CD10.

For conjugates that are designed to be internalized by a cell, group Cpreferably comprises an amino acid sequence selected for cleavage by anendosomal or lysosomal protease, especially the latter. Non-limitingexamples of such proteases include cathepsins B, C, D, H, L and S,especially cathepsin B. Cathepsin B preferentially cleaves peptides at asequence -AA²-AA¹- where AA¹ is a basic or strongly hydrogen bondingamino acid (such as lysine, arginine, or citrulline) and AA² is ahydrophobic amino acid (such as phenylalanine, valine, alanine, leucine,or isoleucine), for example Val-Cit (where Cit denotes citrulline) orVal-Lys. (Herein, amino acid sequences are written in the N-to-Cdirection, as in H₂N-AA²-AA¹-CO₂H, unless the context clearly indicatesotherwise.) For additional information regarding cathepsin-cleavablegroups, see Dubowchik et al., Biorg. Med. Chem. Lett. 8, 3341-3346(1998); Dubowchik et al., Bioorg. Med. Chem. Lett., 8 3347-3352 (1998);and Dubowchik et al., Bioconjugate Chem. 13, 855-869 (2002); thedisclosures of which are incorporated by reference. Another enzyme thatcan be utilized for cleaving peptidyl linkers is legumain, a lysosomalcysteine protease that preferentially cleaves at Ala-Ala-Asn.

In one embodiment, Group C is a peptide comprising the two-amino acidsequence -AA²-AA¹- wherein AA¹ is lysine, arginine, or citrulline andAA² is phenylalanine, valine, alanine, leucine or isoleucine. In anotherembodiment, C consists of a sequence of one to five amino acids,selected from the group consisting of Val-Cit, Ala-Val, Val-Ala-Val,Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Cit-Cit, Val-Lys, Ala-Ala-Asn, Lys,Cit, Ser, and Glu.

The preparation and design of cleavable groups C consisting of a singleamino acid is disclosed in Chen et al., US 2010/0113476 A1, thedisclosure of which is incorporated herein by reference.

Group C can also be a photocleavable one, for example a nitrobenzylether that is cleaved upon exposure to light.

Group C can be bonded directly to antibody Z or compound D; that is,spacers X^(Z) and X^(D), as the case may be, can be absent. For example,if group C is a disulfide, one of the two sulfurs can be a cysteineresidue or its surrogate on antibody Z. Or, group C can be a hydrazonebonded to an aldehyde on a carbohydrate side chain of the antibody. Or,group C can be a peptide bond formed with a lysine ε-amino group ofantibody Z. In a preferred embodiment, compound D is directly bonded togroup C via a peptidyl bond to a carboxyl or amine group in compound D.

When present, spacer X^(Z) provides spatial separation between group Cand antibody Z, lest the former sterically interfere with antigenbinding by latter or the latter sterically interfere with cleavage ofthe former. Further, spacer X^(Z) can be used to confer increasedsolubility or decreased aggregation properties to conjugates. A spacerX^(Z) can comprise one or more modular segments, which can be assembledin any number of combinations. Examples of suitable segments for aspacer X^(Z) are:

where the subscript r is 1 to 24, preferably 2 to 4. These segments canbe combined to make spacers X^(Z) such as:

Spacer X^(D), if present, provides spatial separation between group Cand compound D, lest the latter interfere sterically or electronicallywith cleavage of the former. Spacer X^(D) also can serve to introduceadditional molecular mass and chemical functionality into a conjugate.Generally, the additional mass and functionality will affect the serumhalf-life and other properties of the conjugate. Thus, through judiciousselection of spacer groups, the serum half-live of a conjugate can bemodulated. Spacer X^(D) also can be assembled from modular segments, asdescribed above in the context of spacer X^(Z).

Either spacer X^(Z) or X^(D), or both, can comprise a self-immolatingmoiety. A self-immolating moiety is a moiety that (1) is bonded to groupC and either antibody Z or cytotoxin D and (2) has a structure such thatcleavage from group C initiates a reaction sequence resulting in theself-immolating moiety disbonding itself from antibody Z or cytotoxin D,as the case may be. In other words, reaction at a site distal fromantibody Z or cytotoxin D (cleavage from group C) causes the X^(Z)—Z orthe X^(D)-D bond to rupture as well. The presence of a self-immolatingmoiety is desirable in the case of spacer X^(D) because, if, aftercleavage of the conjugate, spacer X^(D) or a portion thereof were toremain attached to cytotoxin D, the biological activity of the lattermay be impaired. The use of a self-immolating moiety is especiallydesirable where cleavable group C is a polypeptide.

Exemplary self-immolating moieties (i)-(v) bonded to a hydroxyl or aminogroup on a partner molecule D are shown below:

The self-immolating moiety is the structure between dotted lines a andb, with adjacent structural features shown to provide context.Self-immolating moieties (i) and (v) are bonded to a compound D-NH₂(i.e., compound D is conjugated via an amino group), whileself-immolating moieties (ii), (iii), and (iv) are bonded to a compoundD-OH (i.e., compound D is conjugated via a hydroxyl or carboxyl group).Cleavage of the amide bond at dotted line b releases the amide nitrogenas an amine nitrogen, initiating a reaction sequence that results in thecleavage of the bond at dotted line a and the consequent release of D-OHor D-NH₂, as the case may be. For additional disclosures regardingself-immolating moieties, see Carl et al., J. Med. Chem., 24 (3),479-480 (1981); Carl et al., WO 81/01145 (1981); Dubowchik et al.,Pharmacology & Therapeutics, 83, 67-123 (1999); Firestone et al., U.S.Pat. No. 6,214,345 B1 (2001); Toki et al., J. Org. Chem. 67, 1866-1872(2002); Doronina et al., Nature Biotechnology 21 (7), 778-784 (2003)(erratum, p. 941); Boyd et al., U.S. Pat. No. 7,691,962 B2; Boyd et al.,US 2008/0279868 A1; Sufi et al., WO 2008/083312 A2; Feng, U.S. Pat. No.7,375,078 B2; and Senter et al., US 2003/0096743 A1; the disclosures ofwhich are incorporated by reference.

Compound D—Linker Compositions

Conjugates of this invention preferably are prepared by first joining acompound D and linker (X^(D))_(a)C(X^(Z))_(b) to form a drug-linkercomposition represented by formula (V-a):

D-(X^(D))_(a)C(X^(Z))_(b)—R³¹  (V-a)

where R³¹ is a functional group suitable for reacting with a functionalgroup on antibody Z to form the conjugate. Examples of suitable groupsR³¹ include:

where R³² is Cl, Br, F, mesylate, or tosylate and R³³ is Cl, Br, I, F,OH, —O—N-succinimidyl, —O-(4-nitrophenyl), —O-pentafluorophenyl, or—O-tetrafluorophenyl. Chemistry generally usable for the preparation ofsuitable moieties D-(X^(D))_(a)C(X^(Z))_(b)—R³¹ is disclosed in Ng etal., U.S. Pat. No. 7,087,600 B2 (2006); Ng et al., U.S. Pat. No.6,989,452 B2 (2006); Ng et al., U.S. Pat. No. 7,129,261 B2 (2006); Ng etal., WO 02/096910 A1; Boyd et al., U.S. Pat. No. 7,691,962 B2; Chen etal., US 2006/0004081 A1; Gangwar et al., US 2006/0247295 A1; Boyd etal., US 2008/0279868 A1; Gangwar et al., US 2008/0281102 A1; Gangwar etal., US 2008/0293800 A1; Sufi et al., WO 2008/083312 A2; and Chen etal., US 2010/0113476 A1; the disclosures of which are incorporatedherein by reference.

In a preferred embodiment, R³¹ is a maleimide group and the cytotoxiccompound-linker molecule can be represented by formula (V-b):

wherein

-   -   n is 0, 1, or 2;    -   R¹, R² and R³ are independently H, unsubstituted or substituted        C₁-C₁₀ alkyl, unsubstituted or substituted C₂-C₁₀ alkenyl,        unsubstituted or substituted C₂-C₁₀ alkynyl, unsubstituted or        substituted aryl, unsubstituted or substituted heteroaryl,        unsubstituted or substituted (CH₂)₁₋₂O(C₁-C₁₀ alkyl),        unsubstituted or substituted (CH₂)₁₋₂O(C₂-C₁₀ alkenyl),        unsubstituted or substituted (CH₂)₁₋₂O(C₂-C₁₀ alkynyl),        (CH₂)₁₋₂OC(═O)(C₁-C₁₀ alkyl), unsubstituted or substituted        (CH₂)₁₋₂OC(═O)(C₂-C₁₀ alkenyl), unsubstituted or substituted        (CH₂)₁₋₂OC(═O)(C₂-C₁₀ alkynyl), unsubstituted or substituted        C(═O)(C₁-C₁₀ alkyl), unsubstituted or substituted C(═O)(C₂-C₁₀        alkenyl), unsubstituted or substituted C(═O)(C₂-C₁₀ alkynyl),        unsubstituted or substituted cycloaliphatic, unsubstituted or        substituted heterocycloaliphatic, unsubstituted or substituted        arylalkyl, or unsubstituted or substituted alkylaryl;    -   R^(4′) is

-   -   -   wherein R¹² is H, C₁-C₅ alkyl, C₂-C₅ alkenyl, or C₂-C₅            alkynyl; and

    -   R⁵ is H, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, CO(C₁-C₅        alkyl), CO(C₂-C₅ alkenyl), or CO(C₂-C₅ alkynyl);

    -   X^(D) and X^(Z) are spacer groups;

    -   C is a cleavable group; and

    -   a and b are independently 0 or 1;        wherein group R^(4′) is linked via a carboxyl or amine group        therein to either group X^(D) in the event a is 1 or to group C        in the event a is 0.

Preferably, the link between the carboxyl or amine group in R^(4′) andgroup X^(D) or C, as the case may be, is via an amide bond.

In the structures

the stereochemistry at the methyl group alpha to the carboxyl grouppreferably corresponds to that of the naturally occurring tubulysins,i.e.:

Preferably, in formula (V-b) n is 1, R¹ is an isoleucyl residue, a is 0,b is 1, and C comprises one to five amino acids (preferably one to two),and R^(4′) is bonded to C by a peptidyl bond that is enzymaticallycleavable, and R⁵ is Me. This preferred embodiment is represented byformula (V-c):

where R², R³, and R^(4′) are as defined in respect of formula (V-b),each AA independently is a natural amino acid, and X^(Z) isCH₂CH₂NHC(═O)(CH₂)₂₋₅ or C(═O)(CH₂)₂₋₅. Preferred amino acids AA arelysine, citrulline, alanine, valine, glycine, and phenylalanine.

In formula (V-b), R¹ preferably is H, C₁-C₅ alkyl, C₂-C₅ alkenyl, orC₂-C₅ alkenyl, and is more preferably an isoleucyl residue, that is:

In formulae (V-b) and (V-c), R² preferably is H, C₁-C₅ alkyl, C₂-C₅alkenyl, CH₂O(C₁-C₅ alkyl), CH₂O(C₂-C₅ alkenyl), CH₂O(C═O)(C₁-C₅ alkyl),or CH₂OC(═O)(C₂-C₅ alkenyl); and more preferably is H, Me, Et, CH₂OMe,CH₂OEt, CH₂OC(═O)i-Bu, CH₂OC(═O)n-Pr, CH₂OC(═O)CH═CH₂, or CH₂OC(═O)Me.

In formulae (V-b) and (V-c), R³ preferably is H, C₁-C₅ alkyl, C₂-C₅alkenyl, C(═O)C₁-C₅ alkyl, or C(═O)C₂-C₅ alkenyl; and more preferably isH, Me, Et, or C(═O)Me.

In formulae (V-b) and (V-c), R^(4′) preferably is

with R^(4′) equaling

and R¹² equaling H, Me, or Et being especially preferred.

In formula (V-b), n preferably is 1 and R⁵ preferably is methyl; thatis, the ring in the Mep subunit preferably is an N-methyl piperidinylone.

In another embodiment, this invention provides a compound having astructure represented by formula (V-d)

where R¹³ is Me, n-Pr, CH₂OMe, or CH₂OC(═O)CH₂CH(Me)₂; R¹⁴ is Me orC(═O)Me; and R¹⁵ is H or C₁-C₅ alkyl (preferably H, Me, or Et); R¹⁶ is alysine ((CH₂)₄NH₂) or citrulline ((CH₂)₃NHC(═O)NH₂) side chain group;R¹⁷ is a valine (C(Me)₂) or alanine (Me) side chain group; and p is 0 or1.

Examples of specific cytotoxic compound-linker constructs of thisinvention are shown below in formulae (VI-a) through (VI-t).Compound-linker (VI-n) is especially preferred. They have a maleimidegroup and are ready for conjugation to an antibody via a sulfhydrylgroup thereon, by a procedure such as that described below.

Preparation of Conjugates

The following is an illustrative procedure, based on introduction offree thiol groups into an antibody by reaction of lysine ε-amino groupswith 2-iminothiolane, followed by reaction with a maleimide-containingdrug-linker moiety such as described above. Initially the antibody isbuffer exchanged into 0.1 M phosphate buffer (pH 8.0) containing 50 mMNaCl and 2 mM diethylene triamine pentaacetic acid (DTPA) andconcentrated to 5-10 mg/mL. Thiolation is achieved through addition of2-iminothiolane to the antibody. The amount of 2-iminothiolane to beadded can be determined by a preliminary experiment and varies fromantibody to antibody. In the preliminary experiment, a titration ofincreasing amounts of 2-iminothiolane is added to the antibody, andfollowing incubation with the antibody for 1 h at RT (room temperature,circa 25° C.), the antibody is desalted into 50 mM pH 6.0 HEPES bufferusing a SEPHADEX™ G-25 column and the number of thiol groups introduceddetermined rapidly by reaction with dithiodipyridine (DTDP). Reaction ofthiol groups with DTDP results in liberation of thiopyridine, which canbe monitored spectroscopically at 324 nm. Samples at a proteinconcentration of 0.5-1.0 mg/mL are typically used. The absorbance at 280nm can be used to accurately determine the concentration of protein inthe samples, and then an aliquot of each sample (0.9 mL) is incubatedwith 0.1 mL DTDP (5 mM stock solution in ethanol) for 10 min at RT.Blank samples of buffer alone plus DTDP are also incubated alongside.After 10 min, absorbance at 324 nm is measured and the number of thiolgroups is quantitated using an extinction coefficient for thiopyridineof 19,800 M⁻¹.

Typically a thiolation level of about three thiol groups per antibody isdesirable. For example, with some antibodies this can be achieved byadding a 15-fold molar excess of 2-iminothiolane followed by incubationat RT for 1 h. The antibody is then incubated with 2-iminothiolane atthe desired molar ratio and then desalted into conjugation buffer (50 mMpH 6.0 HEPES buffer containing 5 mM glycine and 2 mM DTPA). Thethiolated material is maintained on ice while the number of thiolsintroduced is quantitated as described above.

After verification of the number of thiols introduced, the drug-linkermoiety is added at a 3-fold molar excess per thiol. The conjugationreaction is allowed to proceed in conjugation buffer also containing afinal concentration of 5% dimethylsulfoxide (DMSO), or similaralternative solvent. Commonly, the drug-linker stock solution isdissolved in 100% DMSO. The stock solution is added directly to thethiolated antibody, which has enough DMSO added to bring the finalconcentration to 10%, or pre-diluted in conjugation buffer containing afinal concentration of 10% DMSO, followed by addition to an equal volumeof thiolated antibody.

The conjugation reaction mixture is incubated at RT for 2 h withstirring. Following incubation, the conjugation reaction mixture iscentrifuged and filtered through a 0.2 μm filter. Purification of theconjugate can be achieved through chromatography using a number ofmethods. In one method, the conjugate is purified using size-exclusionchromatography on a SEPHACRYL™ 5200 column pre-equilibrated with 50 mMpH 7.2 HEPES buffer containing 5 mM glycine and 50 mM NaCl.Chromatography is carried out at a linear flow rate of 28 cm/h.Fractions containing conjugate are collected, pooled and concentrated.In an alternative method, purification can be achieved throughion-exchange chromatography. Conditions vary from antibody to antibodyand should to be optimized in each case. For example, antibody-drugconjugate reaction mix is applied to an SP-SEPHAROSE™ columnpre-equilibrated in 50 mM pH 5.5 HEPES containing 5 mM glycine. Theantibody conjugate is eluted using a gradient of 0-1 M NaCl inequilibration buffer at pH 5.5. Relevant fractions containing theconjugate are pooled and dialyzed against formulation buffer (50 mM pH7.2 HEPES buffer containing 5 mM glycine and 100 mM NaCl).

Those skilled in the art will understand that the above-describedconditions and methodology are exemplary and non-limiting and that otherapproaches for conjugation are known in the art and usable in thepresent invention.

Using the foregoing techniques, compounds of this invention wereconjugated using antibodies 2A10, an anti-PSMA antibody (Huang et al.,US 2009/0297438); 2H5, an anti-CD70 antibody (Terrett et al., US2009/0028872); 1F4, an anti-CD70 antibody (Coccia et al., WO2008/074004); or 6A4, an anti-mesothelin antibody (Terrett et al., WO2009/045957). The resulting conugates may be depicted by the followingformulae, where Ab represents an antibody. Those skilled in the art willunderstand that in these formulae the cytotoxin-antibody compound ratiois shown as 1:1 for simplicity, but that in actuality the ratio isusually greater, such as between 2 to 3.

Pharmaceutical Compositions

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising a compound of the present disclosure formulatedtogether with a pharmaceutically acceptable excipient. It may optionallycontain one or more additional pharmaceutically active ingredients, suchas an antibody or another drug. The pharmaceutical compositions can beadministered in a combination therapy with another therapeutic agent,especially another anti-cancer agent.

The pharmaceutical composition may comprise one or more excipients.Excipients that may be used include carriers, surface active agents,thickening or emulsifying agents, solid binders, dispersion orsuspension aids, solubilizers, colorants, flavoring agents, coatings,disintegrating agents, lubricants, sweeteners, preservatives, isotonicagents, and combinations thereof. The selection and use of suitableexcipients is taught in Gennaro, ed., Remington: The Science andPractice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), thedisclosure of which is incorporated herein by reference.

Preferably, a pharmaceutical composition is suitable for intravenous,intramuscular, subcutaneous, parenteral, spinal or epidermaladministration (e.g., by injection or infusion). Depending on the routeof administration, the active compound may be coated in a material toprotect it from the action of acids and other natural conditions thatmay inactivate it. The phrase “parenteral administration” means modes ofadministration other than enteral and topical administration, usually byinjection, and includes, without limitation, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion. Alternatively, the pharmaceuticalcomposition can be administered via a non-parenteral route, such as atopical, epidermal or mucosal route of administration, for example,intranasally, orally, vaginally, rectally, sublingually or topically.

Pharmaceutical compositions can be in the form of sterile aqueoussolutions or dispersions. They can also be formulated in amicroemulsion, liposome, or other ordered structure suitable to achievehigh drug concentration.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated and the particular mode of administration and willgenerally be that amount of the composition which produces a therapeuticeffect. Generally, out of one hundred per cent, this amount will rangefrom about 0.01 per cent to about ninety-nine percent of activeingredient, preferably from about 0.1 per cent to about 70 per cent,most preferably from about 1 per cent to about 30 per cent of activeingredient in combination with a pharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide a therapeutic response. Forexample, a single bolus may be administered, several divided doses maybe administered over time, or the dose may be proportionally reduced orincreased as indicated by the exigencies of the situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. “Dosageunit form” refers to physically discrete units suited as unitary dosagesfor the subjects to be treated; each unit containing a predeterminedquantity of active compound calculated to produce the desiredtherapeutic response, in association with the required pharmaceuticalcarrier.

The dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01to 5 mg/kg, of the host body weight. For example dosages can be 0.3mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kgbody weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.Exemplary treatment regimens are administration once per week, onceevery two weeks, once every three weeks, once every four weeks, once amonth, once every 3 months, or once every three to 6 months. Preferreddosage regimens include 1 mg/kg body weight or 3 mg/kg body weight viaintravenous administration, using one of the following dosing schedules:(i) every four weeks for six dosages, then every three months; (ii)every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kgbody weight every three weeks. In some methods, dosage is adjusted toachieve a plasma antibody concentration of about 1-1000 μg/mL and insome methods about 25-300 μg/mL.

A “therapeutically effective amount” of a compound of the inventionpreferably results in a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction.For example, for the treatment of tumor-bearing subjects, a“therapeutically effective amount” preferably inhibits tumor growth byat least about 20%, more preferably by at least about 40%, even morepreferably by at least about 60%, and still more preferably by at leastabout 80% relative to untreated subjects. A therapeutically effectiveamount of a therapeutic compound can decrease tumor size, or otherwiseameliorate symptoms in a subject, which is typically a human but can beanother mammal.

The pharmaceutical composition can be a controlled or sustained releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See,e.g., Sustained and Controlled Release Drug Delivery Systems, J. R.Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be administered via medical devices such as(1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos.5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and4,596,556); (2) micro-infusion pumps (U.S. Pat. No. 4,487,603); (3)transdermal devices (U.S. Pat. No. 4,486,194); (4) infusion apparati(U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S.Pat. Nos. 4,439,196 and 4,475,196); the disclosures of which areincorporated herein by reference.

In certain embodiments, the pharmaceutical composition can be formulatedto ensure proper distribution in vivo. For example, to ensure that thetherapeutic compounds of the invention cross the blood-brain barrier,they can be formulated in liposomes, which may additionally comprisetargeting moieties to enhance selective transport to specific cells ororgans. See, e.g. U.S. Pat. Nos. 4,522,811; 5,374,548; 5,416,016; and5,399,331; V.V. Ranade (1989) J. Clin. Pharmacol. 29:685; Umezawa etal., (1988) Biochem. Biophys. Res. Commun. 153:1038; Bloeman et al.(1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. AgentsChemother. 39:180; Briscoe et al. (1995) Am. J. Physiol. 1233:134;Schreier et al. (1994) J. Biol. Chem. 269:9090; Keinanen and Laukkanen(1994) FEBS Lett. 346:123; and Killion and Fidler (1994) Immunomethods4:273.

Uses

Compounds of this invention or their conjugatescan be used for treatingdiseases such as, but not limited to, hyperproliferative diseases,including: cancers of the head and neck which include tumors of thehead, neck, nasal cavity, paranasal sinuses, nasopharynx, oral cavity,oropharynx, larynx, hypopharynx, salivary glands, and paragangliomas;cancers of the liver and biliary tree, particularly hepatocellularcarcinoma; intestinal cancers, particularly colorectal cancer; ovariancancer; small cell and non-small cell lung cancer (SCLC and NSCLC);breast cancer sarcomas, such as fibrosarcoma, malignant fibroushistiocytoma, embryonal rhabdomyosarcoma, leiomysosarcoma,neurofibrosarcoma, osteosarcoma, synovial sarcoma, liposarcoma, andalveolar soft part sarcoma; leukemias such as acute promyelocyticleukemia (APL), acute myelogenous leukemia (AML), acute lymphoblasticleukemia (ALL), and chronic myelogenous leukemia (CML); neoplasms of thecentral nervous systems, particularly brain cancer; multiple myeloma(MM), lymphomas such as Hodgkin's lymphoma, lymphoplasmacytoid lymphoma,follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, mantlecell lymphoma, B-lineage large cell lymphoma, Burkitt's lymphoma, andT-cell anaplastic large cell lymphoma. Clinically, practice of themethods and use of compositions described herein will result in areduction in the size or number of the cancerous growth and/or areduction in associated symptoms (where applicable). Pathologically,practice of the method and use of compositions described herein willproduce a pathologically relevant response, such as: inhibition ofcancer cell proliferation, reduction in the size of the cancer or tumor,prevention of further metastasis, and inhibition of tumor angiogenesis.The method of treating such diseases comprises administering atherapeutically effective amount of an inventive combination to asubject. The method may be repeated as necessary. Especially, the cancercan be colorectal cancer, liver cancer, prostate cancer, breast cancer,melanoma, glioblastoma, lung cancer, pancreatic cancer, ovarian cancer,multiple myeloma, renal cancer, leukemia (especially ALL, APL, or AML),or lymphoma.

Compounds of this invention or their conjugates can be administered incombination with other anti-cancer or cytotoxic agents, includingantibodies, alkylating agents, angiogenesis inhibitors, antimetabolites,DNA cleavers, DNA crosslinkers, DNA intercalators, DNA minor groovebinders, enediynes, heat shock protein 90 inhibitors, histonedeacetylase inhibitors, immunomodulators, microtubule stabilizers,nucleoside (purine or pyrimidine) analogs, nuclear export inhibitors,proteasome inhibitors, topoisomerase (I or II) inhibitors, tyrosinekinase inhibitors, and serine/threonine kinase inhibitors. Specificanti-cancer or cytotoxic agents include β-lapachone, ansamitocin P3,auristatin, bicalutamide, bleomycin, bortezomib, busulfan, callistatinA, camptothecin, capecitabine, CC-1065, cisplatin, cryptophycins,daunorubicin, disorazole, docetaxel, doxorubicin, duocarmycin, dynemycinA, epothilones, etoposide, floxuridine, floxuridine, fludarabine,fluoruracil, gefitinib, geldanamycin,17-allylamino-17-demethoxygeldanamycin (17-AAG),17-(2-dimethylaminoethyl)amino17-demethoxygeldanamycin (17-DMAG),gemcitabine, hydroxyurea, imatinib, interferons, interleukins,irinotecan, maytansine, methotrexate, mitomycin C, oxaliplatin,paclitaxel, suberoylanilide hydroxamic acid (SAHA), thiotepa, topotecan,trichostatin A, vinblastine, vincristine, vindesine, lenalidomide(REVLIMID®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), andcetuximab (ERBITUX®).

EXAMPLES

The practice of this invention can be further understood by reference tothe following examples, which are provided by way of illustration andnot of limitation.

Example 1 Scheme 1

Scheme 1 (FIGS. 1 a and 1 b) depicts a method for making compounds ofthis invention.

Thioamide 2

2,2-Diethoxyacetonitrile 1 (25 g, 193 mmol) was mixed with (NH₄)₂S (40mL, 265 mmol, 45% aq. solution in) in 300 mL of methanol (MeOH) at roomtemperature (RT). After keeping the reaction mixture overnight, it wasconcentrated under vacuum and the residue was taken up in ethyl acetate(EtOAc). The EtOAc solution was washed with saturated NaHCO₃ solutionthen brine and dried over anhydrous Na₂SO₄. The EtOAc was evaporated togive thioamide 2 (26 g, 159 mmol, 82%) as a white solid. ¹H NMR (400MHz, CDCl₃) δ 5.01 (s, 1H), 3.67 (m, 4H), 1.22 (t, J=7.2 Hz, 6H).

Methyl 2-(diethoxymethyl)thiazole-4-carboxylate 3

100 g of molecular sieves (3A) was added to a reaction mixture ofthioamide 2 (25 g, 153 mmol) and methyl bromopyruvate (20 mL, 163 mmol)in 300 mL of MeOH. After the mixture was refluxed for 1.5 h, it wascooled and filtered through CELITE™. The filtrate was concentrated andpassed through a column (dichloromethane (DCM):EtOAc, 8:1) to givethiazole carboxylate 3 (34.5 g, 140 mmol, 91%) as a solid, which wasused for next step without further purification.

Methyl 2-formylthiazole-4-carboxylate 4

Thiazole-4-carboxylate 3 (30 g, 122 mmol) was dissolved in 300 mL ofacetone, to which was added aqueous HCl (21 mL, 2M). After keeping thereaction mixture at RT for overnight, the reaction mixture was heated upand kept at 60° C. for 2 h. The reaction mixture was then cooled andevaporated under vacuum to give a residue which was taken up in 200 mLof DCM. DCM solution was then washed with saturated NaHCO₃ solution andthen brine and dried over anhydrous Na₂SO₄. DCM solution was filteredand concentrated under vacuum to give the concentrated solution whichwas triturated by ether to give methyl 2-formylthiazole-4-carboxylate 4(14 g, 82 mmol, 54% for two steps) as a white solid. ¹HNMR (400 MHz,CDCl₃) δ 133-8-p 110.16 (d, J=1.3 Hz, 1H), 8.53 (d, J=1.3 Hz, 1H), 4.01(s, 3H).

Sulfinimine 7

(S)-2-Methylpropane-2-sulfinamide 5 (7.3 mL, 68 mmol) was dissolved in100 mL tetrahydrofuran (THF), to which was added Ti(OEt)₄ (27 mL, 130mmol) and 3-methyl-2-butanone 6 (8 g, 41 mmol) at RT. The reactionmixture was refluxed overnight and then cooled and added to a brinesolution. The resultant mixture was filtered and the cake was washedwith EtOAc. The organic phase was concentrated to give a residue whichwas subjected silica gel column chromatography (DCM:EtOAc, 4:1) to givesulfinimine 7 (9.5 g, 37 mmol, 75%) as an oil. ¹HNMR (400 MHz, CDCl₃) δ2.53 (m, 1H), 2.29 (s, 3H), 1.22 (s, 9H), 1.12 (d, J=4.2 Hz, 3H), 1.10(d, J=4.2 Hz, 3H) MS (ES+) m/z, calculated: m+1, 190.12. found, 190.

Compound 8

Lithium diisopropyl amide (“LDA,” 60 mL, 108 mmol, 1.8M) was added to200 mL of ether at −78° C. followed by the addition of sulfinimine 7(18.9 g, 100 mmol) in 200 mL ether and the resultant reaction mixturewas stirred for 40 min. ClTi(OiPr)₃ (203 mmol, 48.4 mL) was added to thereaction mixture and the solution was stirred for 60 min. A solution ofmethyl 2-formylthiazole-4-carboxylate 4 (12.5 g, 72.6 mmol) in 180 mL ofTHF was added slowly to the reaction mixture. After another 2 h at −78°C., a mixture of acetic acid and THF (⅕ v/v, 4.9 mL) was added. Themixture was warmed to 5° C. over 1 h and then poured into brinesolution. The desired product was then extracted from the brine solutionwith ether and EtOAc solution. The organic phase was then dried overanhydrous MgSO₄, filtered and evaporated. The residue was passed through2 columns (DCM:EtOAc and hexane:EtOAc) to give compound 8 (19.6 g, 54mmol, 75%) as an oil. MS (ES+) m/z, calculated: m+1, 361.12. found, 361.

Compound 9

A solution of compound 8 (19 g, 52.7 mmol) in 200 mL of THF was cooledto −78° C., after which Ti(OEt)₄ (21.7 mL, 105 mmol) was added slowly.After 60 min, when the solution became clear, NaBH₄ (31 mmol, 1.17 g)was added, after 2 h (longer reaction time caused reduction of theester) 10 mL MeOH was added. The mixture was then warmed to 0° C.,poured into 1 mL HOAc in a lot of ice. The mixture was filtered and thecake was washed with EtOAc. After separation, the organic phase wasdried with Na₂SO₄ and evaporated. The final residue was passed through acolumn (DCM:EtOAc, 1:4) to give compound 9 (19 g, 52 mmol, 99%) as anoil. ¹HNMR (400 MHz, CDCl₃) δ 8.1 (s, 1H), 5.54 (d, J=6.7 Hz, 1H), 5.16(m, 1H), 3.92 (s, 3H), 3.42 ((m, 1H), 3.32 (d, J=8.4 Hz, 1H), 2.25 (m,1H), 1.88 (m, 1H), 1.68 (m, 1H), 1.26 (s, 9H), 0.91 (d, J=6.8 Hz, 3H),0.87 (d, J=6.7 Hz, 3H). ¹³CNMR (100 MHz, CDCl₃) δ 177.9, 162.1, 146.6,127.7, 67.9, 58.6, 56.4, 52.5, 40.8, 33.9, 23.1, 19.8, 17.4. MS (ES+)m/z, calculated: m+1, 363.13. found, 363.

Dimethylated Compound 10

Sodium hydride (9.69 mmol, 60%, 387 mg) was added to a solution ofcompound 9 in 6 mL N,N-dimethylformamide (DMF) at 5° C. followed bymethyl iodide (607 uL, 9.7 mmol) after 60 minutes. After stirring thereaction mixture for 3 h, the mixture was poured into ice-cooledsaturated NH₄Cl solution. Ethyl ether was added and the organic layerwas washed with brine, dried over anhydrous MgSO₄ and concentrated togive a residue. The residue was passed through a column (hexane:EtOAc1:4) to give dimethylated compound 10 (314 mg, 0.805 mmol, 33%) as aliquid. ¹HNMR (400 MHz, CDCl₃) δ 8.17 (s, 1H), 4.87 (dd, J=11.0 Hz,J=2.5 Hz, 1H), 3.94 (s, 3H), 3.50 (s, 3H), 3.40 (m, 1H), 2.58 (s, 3H),1.99 (m, 1H), 1.83 (m, 2H), 1.25 (s, 9H), 0.98 (d, J=6.8 Hz, 3H), 0.95(d, J=6.7 Hz, 3H) MS (ES+) m/z, calculated: m+1, 391.16. found, 391.

Monomethyl Amine 11

Aqueous HCl (4N, in dioxane, 0.5 mL) was added to a solution ofdimethylated compound 10 (370 mg, 0.95 mmol) in 5 mL MeOH. Afterstirring the reaction mixture for 60 min, it was evaporated under vacuumto give monomethyl amine 11 (362 mg) as its HCl salt, which was used fornext step without further purification. MS (ES+) m/z, calculated: m+1,287.14. found, 287.

Amide 12

Monomethyl amine 11 (362 mg, 1.12 mmol), Fmoc compound 22 (prepared perWipf et al. 2007; 1.2 g, 3.38 mmol) and N,N-diisopropylethylamine (DIEA,976 uL, 5.6 mmol) were mixed in 5 mL DMF at RT. After stirring for 24 h,the mixture was concentrated and the residue was dissolved in EtOAc. Theorganic phase was washed with NaHCO₃, brine, dried over anhydrous MgSO₄and concentrated to give a residue. The residue was passed through acolumn (hexane:EtOAt:MeOH 7:3:0.6) to give amide 12 (466 mg, 0.75 mmol,67%) as an oil. (ES+) m/z, calculated: m+1, 622.2. found, 622.

Compound 13

Amide 12 (466 mg, 0.75 mmol) was dissolved in 8 mL DCM containing 5%piperidine at RT. After 1 h, the mixture was evaporated under vacuum andthe residue was passed through a column to give an oil (150 mg) whichwas then mixed with(D)-N-methyl pipecolinic acid 23 (“D-Mep,” preparedper Peltier et al., 2006; 50 mg, 0.35 mmol),N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yOuroniumhexafluorophosphate (“HATU,” 126 mg, 0.33 mmol), DIEA (152 uL, 0.84mmol) in 2 mL DCM. After stirring for 2.5 h, the solvent was evaporatedto give a residue which was dissolved in EtOAc. The organic phase wasthen washed with saturated NaHCO₃, brine, dried over anhydrous MgSO₄ andconcentrated to give a residue. The residue was passed through a column(DCM:MeOH 0-10%) to give compound 13 (99 mg, 0,188 mmol, 25%) as an oil.MS (ES+) m/z, calculated: m+1, 525.3. found, 525.

Acid 14

Compound 13 (99 mg, 0.18 mmol) was dissolved in a 3 mL mixture of MeOHand water (3:1 v:v), to which was added NaOH (370 uL, 0.37 mmol, 1M).After stirring for two h, the reaction mixture was neutralized andconcentrated to give a residue. The residue was passed through a C-18column (water (1% trifluoroacetic acid (“TFA”)):acetonitrile (ACN) (1%TFA), 0-100%) to give acid 14 (78 mg, 0.125 mmol, 69%) as a TFA salt. MS(ES+) m/z, calculated: m+1, 511.29. found, 511.

Compound 15

DIEA (24 uL, 137 μmol) was added to a solution of acid 14 (9 mg, 14.4umol, TFA salt) and HATU (6 mg, 15 umol) in DMF (0.5 mL) at RT. Afterall of acid 14 was activated (monitored by HPLC), tubuphenylalanine(prepared per Peltier et al. 2006; 6.5 mg, 27 umol, HCl salt) was added.After stirring for 20 mins, the reaction mixture was passed through aC-18 column (water (1% TFA):ACN (1% TFA), 0-100%) to give compound 15(2.5 mg, 3 umol, 21%) as a white TFA salt. MS (ES+) m/z, calculated:m+1, 700.4. found, 700.

Compound 16

DIEA (20 uL, 0.11 mmol) was added to a solution of acid 14 (12 mg, 0.019mmol, TFA salt), phenylalanine methyl ester (5.3 mg, 0.024 mmol, as HClsalt) and HATU (11.4 mg, 0.03 mmol) in 0.5 mL DMF. After 30 min, thereaction mixture was passed through a C-18 column (water (1% TFA):ACN(1% TFA) 0-100%) to give compound 16 (4.2 mg, 0.005 mmol, 26%) as awhite TFA salt. MS (ES+) m/z, calculated: m+1, 672.89. found, 672.5.Compound 16 is also referred to as compound (III-c) hereinabove.

Compound 17

DIEA (7 uL, 0.04 mmol) was added to a solution of acid 14 (5 mg, 0.008mmol), norvaline methyl ester (“NVaM,” 2 mg, 0.012 mmol) and HATU (4.5mg, 0.012 mmol) in DMF. After stirring the reaction mixture for 30 minthe crude mixture was passed through a C-18 column (water (1% TFA):ACN(1% TFA) 0-100%) to give compound 17 (1.3 mg, 0.0017 mmol, 21%) as awhite TFA salt. MS (ES+) m/z, calculated: m+1, 624.85. found, 624.5.Compound 17 is also referred to as compound (III-e) hereinabove.

Acid 18

Aqueous NaOH (75 uL, 0.75 mmol, 10M) was added to a solution of compound16 (168 mg, 0.25 mmol) in a mixture of MeOH and THF. After stirringovernight, the mixture was neutralized and lyophilized to dryness. Thesolid was used for next step without further purification. MS (ES+) m/z,calculated: m+1, 658.36. found, 658.4. Acid 18 is also referred to ascompound (III-o) hereinabove.

Norvalyl Amide 19

DIEA (5, uL, 0.03 mmol) was added to a solution of acid 18 (5 mg, 0.006mmol), HATU (3.5 mg, 0.009 mmol) and NVaM (1.5 mg, 0.009 mmol) in DMF.After stirring for 30 min, the reaction mixture was passed through aC-18 column (water (1% TFA):ACN (1% TFA) 0-100%) to give norvalyl amide19 (2.2, 0.0025 mmol, 40%) as a white TFA salt. MS (ES+) m/z,calculated: m+1, 772.02. found, 771.5. Norvalyl amide 19 is alsoreferred to as compound (III-f) hereinabove.

Compounds 24, 25, and 26

These three compounds were synthesized from acid 14 or 18 usingprocedures analogous to those described above. The products were allpurified by a C-18 column (water (1% TFA):ACN (1% TFA) 0-100%). Theyields varied from 25-50%. Compound 24: MS (ES+) m/z, calculated: m+1,743.4. found 743. Compound 25: MS (ES+) m/z, calculated: m+1, 686.39.found 686.5. Compound 26: MS (ES+) m/z, calculated: m+1, 700.40. found700.5.

Compound 24 is also referred to as compound (III-g) hereinabove.

Compound 26a

Compound 14 was coupled with Ala-Phe OMe following the same procedure asdescribed for compound 16. The product was purified by a C-18 column(water (1% TFA):ACN (1% TFA), 0-100%). MS (ES+) m/z, calculated: m+1,743. found 743.4. Compound 26a is also referred to as compound (III-1)hereinabove.

Example 2 Scheme 2

This example describes the synthesis of compounds shown in Scheme 2(FIG. 2).

Compound 28

Phenylalanine methyl ester (10 mg, 46.5 μmol) and HATU (14.7 mg, 38.6umol) were added to a solution of compound 27 (prepared per Peltier etal., 2006; 10 mg, 15.5 μmol, formate salt) in 0.5 mL DMF followed byDIEA at RT. After stirring for 30 minutes, DMSO (2 mL) was added and thereaction mixture was directly subjected to a C-18 column (water (5 mMammonium formate, pH 7.2):ACN 0-100%), to give compound 28 (3.2 mg, 25%)as a white solid (formate salt). MS (ES+) m/z, calculated: m+1, 758.41.found 758.4. Compound 28 is also referred to as compound (III-h)hereinabove.

Compound 29

Acetic anhydride (30 μL, 290 μmol) was added to a solution of compound28 (3.2 mg, 3 μmol, formate salt) in 0.5 mL pyridine at 0° C. Afterstirring for 24 h, solvent was was evaporated from the reaction mixtureand the residue was passed through a regular silica column (DCM:MeOH0-10%) to give compound 29 (2.0 mg, 83%) as an oil. MS (ES+) m/z,calculated: m+1, 800.42. found 800.4. Compound 29 is also referred to ascompound (III-i) hereinabove.

O-Acetyl, N,O-acetal 29a

Compound 29 (2 mg, 2.4 μmol) was dissolved in 0.5 mL MeOH, to which wasadded a drop of 4N HCl in dioxane. After stirring the reaction mixtureovernight at RT, the mixture was concentrated and the residue wasdissolved in DMSO, which was then passed through a C-18 column (water(20 mM ammonium formate, pH 6.1): ACN (0-100%) to give O-acetyl,N,O-acetal 29a (1.38 mg, 75%) as a white solid (formate salt) afterlyophilization. MS (ES+) m/z, calculated: m+1, 730. found 730.4.O-Acetyl, N,O-acetal 29a is also referred to as compound (III-m)hereinabove.

N,O-Acetal 29b

Compound 28 (5 mg, 6 umol) was dissolved in MeOH, to which was added 1drop of 4N HCl in dioxane. After stirring the reaction mixture for 24hrs, the solution was concentrated and used for next step reactionwithout further purification. MS (ES+) m/z, calculated: m+1, 687. found688.4. N,O-Acetal 29b is also referred to as compound (III-n)hereinabove.

O-Methyl, N,O-acetal 29c

N,O-Acetal 29b (about 5 mg, 7.2 umol) was dissolved in DMF, to which wasadded dimethylsulfate (3 μL, 37 μmol) and NaH (2 mg, 50 μmol, 60% inmineral oil) at 0° C. After 1 h, the mixture was taken up in DMSO andpassed through a C-18 column (water (20 mM ammonium formate, pH 6.1):ACN (0-100%) to give O-methyl, N,O-acetal 29c as a semi-solid (0.31 mg,5%; mixture containing an unidentified compound of same MW). MS (ES+)m/z, calculated: m+1, 702. found 702.4. O-methyl, N,O-acetal 29c is alsoreferred to as compound (III-k) hereinabove.

Example 3 Scheme 3

Scheme 3 (FIG. 3) shows a procedure for making compounds according toformula (II-b).

Alcohol 30

TFA (171 mL) was added to a solution of(S)-tert-butyl-1-hydroxy-3-(4-nitrophenyl)propan-2-ylcarbamate 20(Erlanson et al., U.S. Pat. No. 7,214,487 B2; 13.9 g, 46.9 mmol) in DCM(272 mL) at 0° C. The reaction mixture was warmed to RT and reaction wasallowed to proceed for 25 minutes. The solution was concentrated toafford 9.2 g of crude (S)-2-amino-3-(4-nitrophenyl)propan-1-ol as awhite solid. To a solution of this crude product and sodium carbonate(12.4 g, 117.3 mmol) in THF (87 mL) and water (87 mL) was addedN-carbethoxyphthalimide (“CEPT,” 12.3 g, 56.3 mmol). After the reactionmixture was stirred at RT for 4 h, EtOAc (150 mL) was added. The aqueousphase was extracted with EtOAc. The combined organic layers were washedwith brine, dried over anhydrous Na₂SO₄, filtered and concentrated undervacuum to give a crude residue which was purified by flashchromatography eluting from silica gel with a gradient of 0-100% EtOAcin hexanes to give 12.3 g of alcohol 30. MS: (+) m/z 327.0 (M+1).

Triflate 31

To a solution of alcohol 30 (1 g, 3.06 mmol) in anhydrous DCM (18 mL)was added pyridine (0.274 mL, 3.36 mmol) at −78° C. After the reactionmixture was stirred at −78° C. for 5 min, trifluoromethanesulfonicanhydride (0.568 mL, 3.36 mmol) was added. The reaction mixture wasstirred at −78° C. for 45 min, and then at RT for 45 min. After theprecipitate was filtered off, the filtrate was purified by flashchromatography eluting from silica gel with DCM to yield 0.84 g oftriflate 31. ¹H NMR (400 MHz, CDCl₃) δ 8.10 (2H, d, J=8.8 Hz), 7.81 (2H,m), p 7.74 (2H, m), 7.36 (2H, J=8.8 Hz), 5.13 (1H, t, J=10.0 Hz), 4.99(1H, m), 4.80 (1H, dd, J=4.8, 5.6 Hz), 3.52 (1H, dd, J=3.2, 11.2 Hz),and 3.27 (1H, dd, J=5.6, 8.8 Hz).

Diester 32

Diethyl methylmalonate (0.71 mL, 4.12 mmol) was added dropwise to asuspension of sodium hydride (0.161 g, 60% dispersion in mineral oil,4.03 mmol) in anhydrous THF (4.7 mL) at 0° C. The reaction mixture wasstirred at 0° C. for 10 min, and then at RT for 10 min. The resultingsolution was added slowly to a solution of triflate 31 (0.84 g, 1.83mmol), in anhydrous THF (9.4 mL) at 0° C. After the reaction mixture wasstirred at 0° C. overnight, saturated NH₄Cl aqueous solution (20 mL) wasadded. The aqueous solution was extracted with EtOAc, and the combinedorganic layers were washed with brine, dried over MgSO₄, filtered, andconcentrated under vacuum to give a residue. The crude product waspurified by flash chromatography eluting from silica gel with a gradientof 0-50% EtOAc in hexanes to afford 0.57 g of diester 32. MS: (+) m/z483.3 (M+1).

Monoester 33

A solution of diester 32 in 6 N HCl (10 mL) and acetic acid (10 mL) washeated at 145° C. for 2 days. The organic solution was concentrated toafford 0.41 g of the hydrochloride salt of the crude(R)-4-amino-2-methyl-5-(4-nitrophenyl)pentanoic acid as a white solid.

2,2-Dimethoxypropane (“DMP,” 4 mL, 32.6 mmol) was added to a solution ofthe hydrochloride salt of the crude product and concentrated HCl (1 mL)in anhydrous MeOH (20 mL). The reaction mixture was heated at 60° C.overnight. The organic solution was concentrated to afford 0.43 g of thehydrochloride salt of the crude (R)-methyl4-amino-2-methyl-5-(4-nitrophenyl)pentanoate as a white solid.

Triethylamine (0.44 mL, 3.1 mmol) was added to a solution of thehydrochloride salt of the crude (R)-methyl4-amino-2-methyl-5-(4-nitrophenyl)pentanoate and di-tert-butyldicarbonate (0.369 g, 1.69 mmol) in ACN (10 mL) at RT. After thereaction mixture was stirred at RT for 4 h, the solvent was evaporated.Water (20 mL) was added, and the aqueous solution was extracted withEtOAc. The combined organic layers were washed with brine, dried overNa₂SO₄, filtered, and concentrated. The crude product was purified byflash chromatography eluting from silica gel with a gradient of 0-30%EtOAc in hexanes to afford 0.31 g of monoester 33 as a colorless oil.MS: (+) m/z 267.3 (M-99).

Carboxylic Acid 34

A solution of monoester 33 (0.31 g, 0.846 mmol) in 6 N HCl was heated at130° C. for 1.5 h. The organic solution was concentrated to afford 0.244g of carboxylic acid 34 as a white solid. MS: (+) m/z 253.1 (M+1).

Nitro Acid 35

Compound 34a (80.4 mg, 0.149 mmol, prepared per Patterson et al. 2008)was added to a 0.2 M solution of pentafluorophenol (41.1 mg, 0.224 mmol)and N,N′-diisopropylcarbodiimide (“DIC,” 0.0255 mL, 0.164 mmol) in DCM(0.76 mL) at 0° C. The reaction mixture was warmed to RT and stirred atRT overnight. The solvent was evaporated. EtOAc (18 mL) was added andthe crude product was filtered, with rinsing of the reaction vessel withEtOAc. The filtrate was concentrated under reduced pressure and thecrude material was used without further purification. DMF (0.6 mL) wasadded to the crude product, followed by carboxylic acid 34 (0.129 g,0.448 mmol) and DIEA (0.13 mL, 0.745 mmol). The reaction mixture wasstirred at RT overnight and the solvent was evaporated off. The crudeproduct was purified by flash chromatography eluting from silica gelwith a gradient of 10-20% MeOH in DCM containing 1% NH₄OH to afford 0.11g of nitro acid 35 as a white solid. MS: (+) m/z 773.4 (M+1).

Amino Acid 36

A solution of nitro acid 35 (0.11 g, 0.142 mmol) and palladium on carbon(10%, 15 mg) in MeOH (5 mL) was stirred under a hydrogen atmosphere for4 h. The catalyst was filtered off, and the filtrate was concentrated toafford 91 mg of amino acid 36 as a white solid. MS: (+) m/z 743.5 (M+1)Amino acid 36 is also referred to as compound (III-b) hereinabove.

Methyl Ester 36a

HCl (1 drop, 37%) was added to a solution of amino acid 36 (1.9 mg, 2.5mmol) and 2, 2-dimethoxypropane (“DMP,” 0.05 mL, 0.41 mol) in MeOH (0.5mL). The reaction mixture was stirred at RT for 2 h and thenconcentrated. The crude product was purified by preparative HPLC toafford 1.7 mg of methyl ester 36a as a white solid. MS: (+) m/z 757.5(M+1). Ester 36a is also depicted in this specification by formula(III-t).

Example 4 Scheme 4

Scheme 4 (FIG. 4) shows a method for attaching peptidyl linkers andreactive functional groups to compounds of this invention, ready forconjugation.

Compound 37

A solution of DIEA, Fmoc-Lys(Boc)-OH (17.3 mg, 0.037 mmol), and HATU(12.8 mg, 0.0336 mmol) in DMF (0.3 mL) was stirred at RT for 5 min. ThepH of the solution was maintained between 8 and 9. Then a solution ofamino acid 36 (25 mg, 0.0336 mmol) in DMF (2 mL) and DIEA was added tothe reaction mixture, maintaining the pH between 8 and 9. After stirringat RT for 15 min, saturated NH₄Cl solution (5 mL) was added to quenchthe reaction. The aqueous solution was extracted with EtOAc, and thecombined organic layers were dried, filtered, and concentrated. Thecrude product was purified by flash chromatography eluting from silicagel with a gradient of 0-20% MeOH in DCM to afford 36.1 mg of compound37. MS: (+) m/z 1193.6 (M+1).

Compound 38

Piperidine was added to a solution of compound 37 (36.1 mg, 0.0302 mmol)in DMF (2 mL), maintaining pH between 9 and 10. After stirring at RT for20 min, the organic solution was concentrated to afford 29.3 mg of thecrude free α-amino compound.

DIEA was added to a solution of 6-maleimidohexanoic acid (7.0 mg, 0.0332mmol) and HATU (11.5 mg, 0.0302 mmol) in DMF (0.3 mL) maintaining the pHbetween 8 and 9. The reaction mixture was stirred at RT for 5 min. ThenDIEA and the crude free amino compound in DMF (2 mL) were added,maintaining the pH between 8 and 9. After the reaction mixture wasstirred at RT for 15 min, the crude product was purified by preparativeHPLC to afford 9.1 mg of compound 38 as a white solid. MS: (+) m/z1164.6 (M+1).

Compound 39

TFA (1.5 mL) was added to a solution of compound 38 (9.1 mg, 0.0078mmol) in DCM (1.5 mL). After the reaction mixture was stirred at RT for15 min, the crude product was purified by preparative HPLC to afford 5.0mg of the TFA salt of the desired compound 39 as a white solid. MS: (+)m/z 1064.8 (M+1). The free base structure of compound 39 is also shownhereinabove, as compound (VI-b). Some amide of compound 39 was alsoisolated as a by-product in its preparation. MS: (+) m/z 1063.6 (M+1).The amide is also depicted in this specification by formula (VI-q).

Example 5 Scheme 5

Scheme 5 (FIG. 5) shows an alternative procedure for making compoundsaccording to formula (II-b).

Amino Ester 42

4.0 N HCl in 1,4-dioxane (6.7 mL) was added to a solution of compound 41(prepared per Patterson et al. 2008; 1 g, 2.66 mmol) in ethanol (17 mL).The reaction mixture was stirred at RT for 2 h, and then concentrated toafford 0.82 g of amino ester 42 as a white solid. MS: (+) m/z 273.3(M+1).

Azido Ester 43

Oxalyl chloride (1.71 mL, 19.95 mmol) and DMF (0.33 mL, 4.26 mmol) wereadded to a solution of azido isoleucine (Lundquist et al., Org. Lett.2001, 3, 781; 0.669 g, 4.26 mmol) in hexanes (176 mL). The reactionmixture was stirred at RT for 1 h, filtered, and concentrated to affordthe acid chloride. The acid chloride and DIEA (2.32 mL, 13.3 mmol) wereadded to a solution of amino ester 42 (0.82 g, 2.66 mmol) in DCM (26.7mL) at 0° C. The reaction mixture was allowed to warm to RT and stirredat RT overnight. Brine was added to quench the reaction, and the aqueoussolution was extracted with EtOAc. The combined organic layers weredried over Na₂SO₄, filtered, and concentrated under vacuum. The crudeproduct was purified by flash chromatography eluting from silica gelwith a gradient of 0-50% EtOAc in hexanes to afford 0.86 g of azidoester 43 as a white solid. MS: (+) m/z 412.3 (M+1).

Triethylsilyl Compound 44

2,6-Lutidine (1.22 mL, 10.45 mmol) and triethylsilyltrifluoromethanesulfonate (1.14 mL, 5.02 mmol) were added to a solutionof azido ester 43 (0.86 g, 2.09 mmol) in DCM (11 mL) at 0° C. Thereaction mixture was allowed to warm to RT over 1 h, and then stirred atRT for an additional hour. Brine was added to quench the reaction, andthe aqueous solution was extracted with EtOAc. The combined organiclayers were dried, filtered, and concentrated. The crude product waspurified by silica gel flash chromatography eluting with a gradient of0-30% EtOAc in hexanes to afford 1.1 g of triethylsilyl compound 44. MS:(+) m/z 526.4 (M+1).

N-Methyl Compound 45

A solution of triethysilyl compound 44 (1.04 g, 1.98 mmol) in THF (6.5mL) was cooled at −45° C., and potassium hexamethyldisilazide (0.5 M intoluene, 4.75 mL, 2.37 mmol) was added. The resulting mixture wasstirred for 20 min at −45° C. Methyl iodide (0.37 mL, 5.94 mmol) wasadded, and the reaction mixture was allowed to warm to RT over 4 h atwhich time the reaction was quenched with ethanol (10 mL). The crudeproduct was diluted with EtOAc and washed with brine, and the aqueouslayer was extracted with EtOAc. The combined organic layers were driedover anhydrous Na₂SO₄, filtered, and concentrated under vacuum. Thecrude product was purified by flash chromatography eluting from silicagel with a gradient of 0-30% EtOAc in hexanes to afford 0.91 g ofN-methyl compound 45. MS: (+) m/z 540.4 (M+1).

Compound 46

A solution of N-methyl compound 45 (1.0 g, 1.85 mmol) in deoxygenatedAcOH/H₂O/THF (65 mL, 3:1:1, v/v/v) was stirred at RT for 36 h. Toluene(250 mL) was added and the solution was concentrated. The crude productwas purified by flash chromatography eluting from silica gel with agradient of 0-100% EtOAc in hexanes to afford 0.46 g of compound 46 asoil. MS: (+) m/z 426.3 (M+1).

Methyl Ether 47

Potassium hexamethyldisilazide (“KHMDS,” 0.5 M in toluene, 2.54 mL, 1.27mmol) was added to a solution of compound 46 (0.45 g, 1.06 mmol) in THF(5 mL) at −78° C. The reaction mixture was stirred for 20 min at −78° C.Methyl iodide (0.2 mL, 3.18 mmol) was added, and the reaction mixturewas allowed to warm to −20° C. over 2 h at which time the reaction wasquenched with saturated NH₄Cl solution. The aqueous solution wasextracted with EtOAc. The combined organic layers were dried overanhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crudeproduct was purified by flash chromatography eluting from silica gelwith a gradient of 0-50% EtOAc in hexanes to afford 0.41 g of compound47 as colorless oil. MS: (+) m/z 440.3 (M+1).

Compound 48

To a solution of D-Mep (0.45 g, 3.14 mmol) in EtOAc (10 mL) were addedpentafluorophenol (0.64 g, 3.47 mmol) and N,N′-dicyclohexylcarbodiimide(“DCC,” 0.72 g, 3.47 mmol). After the reaction mixture was stirred at RTovernight, the precipitate was filtered, and washed with EtOAc. To theresulting filtrate waa added compound 47 (0.46 g, 1.05 mmol) andpalladium on carbon (10 wt %, 0.36 g). The reaction mixture was stirredunder a hydrogen atmosphere overnight. The catalyst was filtered off,and then the filtrate was concentrated under vacuum. The crude productwas purified by flash chromatography eluting from silica gel with agradient of 0-5% MeOH in EtOAc to afford 0.43 g of compound 48 ascolorless oil. MS: (+) m/z 539.4 (M+1).

Carboxylic acid 49

To a solution of compound 48 (0.43 g, 0.80 mmol) in deoxygenated1,4-dioxane (8 mL) was added deoxygenated lithium hydroxide aqueoussolution (0.6 M, 4 mL) at RT. The reaction mixture was stirred at RT for2 h, and then concentrated under vacuum. The crude product was purifiedby flash chromatography eluting from silica gel with a gradient of10-30% MeOH in DCM containing 1% NH₄OH to afford 0.3 g of carboxylicacid 49 as a white solid. MS: (+) m/z 511.4 (M+1).

Nitro Acid 50

Carboxylic acid 49 (80 mg, 0.157 mmol) was added to a 0.2 M solution ofpentafluorophenol (43.3 mg, 0.235 mmol) and DIC (0.0269 mL, 0.173 mmol)in DCM (0.8 mL) at 0° C. The reaction mixture was warmed to RT, andstirred at such temperature overnight. The solvent was evaporated. Ethylacetate (18 mL) was added, and the crude product was filtered, withEtOAc rinse of the reaction vessel. The filtrate was concentrated underreduced pressure, and the crude material was used without furtherpurification. DMF (0.6 mL) was added to the crude product, followed bycarboxylic acid 34 (0.136 g, 0.47 mmol), and DIEA (0.137 mL, 0.785mmol). The reaction mixture was stirred at RT overnight, and the solventwas then evaporated under vacuum. The crude product was purified byflash chromatography, eluting from silica gel with a gradient of 10-20%MeOH in DCM containing 1% NH₄OH to afford 0.1 g of nitro acid 50 as awhite solid. MS: (+) m/z 745.4 (M+1).

Amino Acid 51

A mixture of nitro acid 50 (0.1 g, 0.134 mmol) and palladium on carbon(10%, 14 mg) in MeOH (5 mL) was stirred under a hydrogen atmosphere for4 h. The catalyst was filtered off, and the filtrate was concentratedunder vacuum to afford 87.3 mg of amino acid 51 as a white solid. MS:(+) m/z 715.5 (M+1) Amino acid 51 is also referred to as compound(III-j) hereinabove.

Example 6 Scheme 6

Scheme 6 (FIG. 6) shows yet another procedure for making compoundsaccording to formula (II-b).

Hydroxy Nitro Compound 52

Compound 27 (Scheme 2) (16.4 mg, 0.0275 mmol), was added to a 0.2 Msolution of pentafluorophenol (7.6 mg, 0.0413 mmol) and DIC (0.0094 mL,0.0606 mmol) in DCM (0.2 mL) at 0° C. The reaction mixture was warmed toRT, and stirred at RT overnight. The solvent was evaporated. EtOAc (3mL) was added and the crude product was filtered, with rinsing of thereaction vessel with EtOAc. The filtrate was concentrated under reducedpressure, and the crude material was used without further purification.DMF (0.1 mL) was added to the crude product, followed by carboxylic acid34 (Scheme 3) (20.8 mg, 0.083 mmol), and N,N-diisopropylethylamine(0.024 mL, 0.138 mmol). The reaction mixture was stirred at RTovernight, and the solvent was then evaporated. The crude product waspurified by flash chromatography eluting from silica gel with a gradientof 0-10% MeOH in DCM to afford 14.9 mg of hydroxy nitro compound 52 as awhite solid. MS: (+) m/z 831.5 (M+1).

Acetyl Nitro Compound 53

A 0.1 M solution of hydroxy nitro compound 52 (14.9 mg, 0.018 mmol) inpyridine (0.23 mL) was cooled at 0° C., and acetic anhydride (0.054 mL,0.57 mmol) was added. The reaction mixture was allowed to warm to RTover 2 h, and stirred at RT for 24 h. The reaction mixture was cooled to0° C., and 1:1 mixture of 1,4-dioxane and water was added. The reactionmixture was allowed to warm to RT, followed by stirring at thistemperature for 12 h. The solvent was evaporated, and the residue waspurified by preparative HPLC to afford 2.2 mg of acetyl nitro compound53 as a white solid. MS: (+) m/z 873.2 (M+1).

Acetyl Amino Compound 54

A mixture of acetyl nitro compound 53 (2.2 mg, 0.0025 mmol) andpalladium on carbon (10%, 1 mg) in methanol (0.2 mL) was stirred under ahydrogen atmosphere for 4 h. The catalyst was filtered off, and thefiltrate was concentrated. The crude product was purified by preparativeHPLC to afford 0.1 mg of acetyl nitro compound 54 as a white solid. MS:(+) m/z 843.2 (M+1). Acetyl amino compound 54 is also referred to ascompound (III-a) hereinabove.

Example 7 Scheme 7

Scheme 7 (FIG. 7) shows yet another procedure for making compounds ofthis invention.

Compound 55

Compound 34a (Scheme 3) (70 mg, 0.13 mmol) was added to a 0.2 M solutionof pentafluorophenol (35.9 mg, 0.195 mmol) and DIC (0.0223 mL, 0.143mmol) in DCM (0.66 mL) at 0° C. The reaction mixture was warmed to RTand stirred at RT overnight. The solvent was evaporated. EtOAc(16 mL)was added and the crude product was filtered, with rinsing of thereaction vessel with EtOAc. The filtrate was concentrated under reducedpressure, and the crude material was used without further purification.DMF (0.5 mL) was added to the crude product, followed byp-nitrophenylalanine (82.0 mg. 0.39 mmol) and DIEA (0.114 mL, 0.65mmol). The reaction mixture was stirred at RTovernight, and the solventwas then evaporated. The crude product was purified by flashchromatography eluting from silica gel with a gradient of 10-20% MeOH inDCM containing 1% NH₄OH to afford 65.2 mg of compound 55 as a whitesolid. MS: (+) m/z 731.0 (M+1).

Compound 56

A mixture of compound 55 (65.2 mg, 0.089 mmol) and palladium on carbon(10%, 9.4 mg) in MeOH (3 mL) was stirred under a hydrogen atmosphere for4 h. The catalyst was filtered off, and the filtrate was concentrated toafford 33.8 mg of compund 56 as a white solid. MS: (+) m/z 701.2 (M+1).Compound 56 is also referred to as compound (III-d) hereinabove.

Example 8 Scheme 8

Scheme 8 (FIG. 8 a) shows a method for making a compound according toformula (VIII-b), useful as an intermediate for making compounds of thisinvention.

Boc Ester 58

To a solution of amino ester 57 (Chem-Impex, 5 g, 19.18 mmol) anddi-tert-butyl dicarbonate (“(Boc)₂O,” Aldrich, 4.6 g, 21.10 mmol) in DMF(Acros, anhydrous, 50 mL), triethylamine (“TEA,” Aldrich, 8.36 mL, 60mmol) was added. The reaction mixture was stirred for 0.5 h. HPLCanalysis showed reaction was completed. The reaction mixture was dilutedwith EtOAc (500 mL) and the organic layer was washed with water (200 mL)and then brine (200 mL), dried over anhydrous MgSO₄ and concentrated.The crude product was purified on 120 g CombiFlash column with 0-5% MeOHin DCM to yield white solid Boc ester 58 (5.6 g, 81%). ¹HNMR (DMSO) δ8.18 (d, 2H), 7.47 (d, 2H), 7.38 (d, 1H), 4.23 (m, 1H), 3.60 (s, 3H),3.15 (m, 1H), 2.95 (m, 1H), 1.23 (s, 9H).

Alkene 59

To a solution of Boc ester 58 (230 mg, 0.68 mmol) in DCM (Acros,anhydrous, 2 mL) cooled to −78° C. in a dry ice-acetone, DIBAL (Aldrich,1 M in DCM, 1 mL) was added slowly. The reaction mixture was stirred andwarmed up to −20° C. over 3 h.(1-Ethoxycarbonylethyliden)-triphenylphosphoran (Aldrich, 492 mg, 1.36mmol) was added. The reaction mixture was stirred at −20° C. for 1 h.The reaction mixture was diluted with EtOAc (100 mL) and the resultantorganics was washed with water (50 mL) and then brine (50 mL), driedover anhydrous MgSO₄ and concentrated. The crude product was purified on10 g COMBIFLASH™ column with 0-50% EtOAc in hexane to yield white solidalkene 59 (151 mg, 59%). ¹HNMR (DMSO) δ 8.18 (d, 2H), 7.47 (d, 2H), 7.22(d, 1H), 6.51 (d, 1H), 4.48 (m, 1H), 4.11 (q, 2H), 2.80-2.94 (m, 2H),1.62 (s, 3H), 1.23 (s, 9H), 1.16 (t, 3H).

Aryl Amine 60

A solution of alkene 59 (148 mg, 0.39 mmol) in EtOH (Acros, anhydrous, 3mL) and Pd on charcoal (Aldrich, 10%, 50 mg) was stirred under H₂overnight. The reaction mixture was diluted with DCM (10 mL) andfiltered through CELITE™. The filtrate was concentrated and the crudeproduct was purified on 4 g COMBIFLASH™ column with 0-20% MeOH in DCM toyield aryl amine 60 as a white solid (102 mg, 75%). ¹HNMR (DMSO) δ 7.18(d, 2H), 7.11 (s, 2H), 6.71 (d, 1H), 3.98 (q, 2H), 3.51 (m, 1H), 2.57(m, 2H), 2.41 (m, 1H), 1.63 (m, 1H), 1.37 (m, 1H), 1.29 (s, 9H), 1.09(t, 3H), 0.99 (d, 3H), MS (ES+) m/z, calculated: m+1, 351.2. found351.2.

Example 9 Scheme 9

Scheme 9 (FIG. 8 b) shows another method for making a compound accordingto formula (VIII-b), useful as an intermediate for making compounds ofthis invention.

Amino Acid 61

A mixture of carboxylic acid 24 (Scheme 3, FIG. 3) (4.4 mg, 0.0025 mmol)and palladium on carbon (10%, 1 mg) in MeOH (0.5 mL) was stirred under ahydrogen atmosphere overnight. The catalyst was filtered off, and thefiltrate was concentrated to afford 3.5 mg of amino acid 61 as a whitesolid. MS: (+) m/z 223.3 (M+1).

Example 10 Schemes 10, 11, and 12

Scheme 10 (FIG. 8 c) shows another method for making a compoundaccording to formula (VIII-b), useful as an intermediate for makingcompounds of this invention.

Compound 62

A mixture of monoester 33 (Scheme 3, FIG. 3) (0.34 g, 0.93 mmol) andpalladium on carbon (10%, 50 mg) in methanol (20 mL) was stirred under ahydrogen atmosphere overnight. The catalyst was filtered off, and thefiltrate was concentrated to afford 0.29 g of compound 62 as a whitesolid. MS: (+) m/z 237.3 (without Boc).

Scheme 11 (FIG. 9) illustrates how compounds according to formula(VIII-b) can be used to make compounds of this invention. Boc ester 62is converted to Bpoc ester 62a by first protecting the aromatic aminegroup with an Fmoc group, treatment with TFA to remove the Boc groupfrom the aliphatic amine group, treatment with carbonic acidα,α-dimethyl-p-phenylbenzyl phenyl ester (8C1) to install a Bpoc groupthere, and removal of the Fmoc group with piperidine. Bpoc ester 62a iscoupled with carboxylic acid 63 with HATU to yield an intermediate esterthat is then hydrolyzed with LiOH to produce compound 64. Hydrogenationto remove the Cbz protecting group from compound 64, followed byHATU-mediated coupling with 6-maleimidohexanoic acid and removal of theBpoc group with acetic acid yields amino acid 65. Another HATU-mediatedcoupling with compound 34a (Scheme 3, FIG. 3) yields compound 66.Removal of the Boc protecting group with TFA affords compound 67, readyfor conjugation.

Yet another mode of utilizing compounds of formula (VIII-b) is shown inScheme 12 (FIG. 10). Starting from compound 34a, HATU-mediated couplingwith compound 68 affords Boc ester 69. Removal of the Boc group with TFAand hydrolysis of the ester with LiOH affords amino acid 36, which canbe elaborated as shown in Scheme 4 (FIG. 4) to prepare a compositionsuitable for conjugation.

Example 11 Compound 70 Compound 70

Tubulysin D (made per Peltier et al. 2006; 2 mg, 2.4 umol) was dissolvedin MeOH at 0° C. To this solution was added a drop of HCl (0.1 M). Afterstirring the reaction mixture for overnight at RT the solution wasevaporated under vacuum to give a residue that was passed through ashort column (DCM:MeOH 0-10%) to give compound 70 (1.3 mg, 1.6 umol,67%) as an oil. MS (ES+) m/z, calculated: m+1, 772.42. found, 772.

Those skilled in the art will appreciate that the general methodology ofthe Schemes can be adapted to make compounds of this invention otherthan those specifically described above. For example, compound 14 (sameas compound 49) can be used to make numerous other compounds of thisinvention by coupling it with other replacements for the Tup subunit. Asanother example, by changing the reagents used with compound 9 (Scheme1), compounds 44 and 46 (Scheme 5), variations on groups R² and R³ informula (II) beyond those specifically exemplified can be synthesized.

Example 12 Preparation of a Conjugate

This example describes the preparation of a conjugate ofcytotoxin-linker construct (VI-b) and anti-CD70 monoclonal antibody 2H5(Terrett et al., US 2009/0028872 A1; Coccia et al., WO 2008/074004 A2).It is representative of the procedure used in the preparation of otherconjugates.

Anti-CD70 antibody 2H5 at ˜5 mg/mL in 20 mM sodium phosphate, 50 mMNaCl, 100 μM DTPA, pH 7.5, was thiolated with a 13-fold molar excess of2-iminothiolane. The thiolation reaction was allowed to proceed for 1 hat RT with continuous mixing.

Following thiolation, the antibody was buffer exchanged into conjugationbuffer (50 mM HEPES, 5 mM glycine, 2 mM DTPA, pH 6.0) via a PD10 column(Sephadex G-25). The concentration of the thiolated antibody wasdetermined at 280 nm. The thiol concentration was measured using adithiodipyridine assay.

A 5 mM stock of construct (VI-b) in DMSO was added at a 3-fold molarexcess per thiol of antibody and mixed for 90 min at RT. Followingconjugation, 100 mM N-ethylmaleimide in DMSO was added at a 10-foldmolar excess of thiol per antibody to quench any unreacted thiol groups.This quenching reaction was done for one h at RT with continuous mixing.

The anti-CD70 antibody drug conjugate was 0.2 μm filtered prior tocation-exchange chromatographic purification. The SP SEPHAROSE™ HighPerformance Cation Exchange column (CEX) was regenerated with 5 columnvolumes (CVs) of 50 mM HEPES, 5 mM glycine, 1M NaCl, pH 6.0. Followingregeneration, the column was equilibrated with 3 CVs of equilibrationbuffer (50 mM HEPES, 5 mM glycine, pH 6.0). The conjugate was loaded andthe column and was washed once with the equilibration buffer. Theconjugate was eluted with 50 mM HEPES, 5 mM glycine, 200 mM NaCl, pH6.0. The eluate was collected in fractions. The column was thenregenerated with 50 mM HEPES, 5 mM glycine, 1M NaCl, pH 6.0 to removeprotein aggregates and any unreacted (VI-b).

Fractions containing monomeric antibody conjugate were pooled. Antibodyconjugate concentration and substitution ratios were determined bymeasuring absorbance at 280 and 252 nm.

The purified eluate pool was buffer exchanged into 30 mg/mL sucrose, 10mg/mL glycine, pH 6.0, by dialysis. Dextran 40 was added to the sampleat 10 mg/mL post-dialysis. The concentration and substitution ratio (SR)were determined by measuring absorbance at 280 and 252 nm. The SR was2.2 moles of cytotoxin per mole of antibody.

Example 13 Proliferation Assays

This example generally describes the procedures used to assay theantiproliferative activity of compounds of this invention or theirconjugates. Human tumor cell lines were obtained from the American TypeCulture Collection (ATCC), P.O. Box 1549, Manassas, Va. 20108, USA, andcultured according to instruction from the ATCC. Cells were seeded at1.0×10³ or 1.0×10⁴ cells/well in 96-well plates for 3 h for ATP assaysor ³H thymidine assays, respectively. 1:3 serial dilutions of free(unconjugated) compounds or their conjugates were added to the wells.Plates were allowed to incubate for 24 to 72 h. The ³H thymidine plateswere pulsed with 1.0 μCi of ³H-thymidine per well for the last 24 hoursof the total incubation period, harvested, and read on a Top CountScintillation Counter (Packard Instruments, Meriden, Conn.). ATP levelsin the ATP plates were measured using the CELLTITER-GLO® LuminescentCell Viability kit following the manufacturer's manual and read on aGLOMAX® 20/20 luminometer (both from Promega, Madison, Wis., USA). TheEC₅₀ values—the concentration at which an agent inhibits or reduces cellproliferation by 50%—were determined using PRISM™ software, version 4.0(GraphPad Software, La Jolla, Calif., USA).

Example 14 Cytotoxin In Vitro Activity

Using the ³H thymidine or the ATP luminescence assay, or both, theactivities of compounds of this invention were assayed against thefollowing cancer cell lines: HCT-15 (colorectal cancer, multi-drugresistant (MDR)); Hep3B (liver cancer); LNCaP (prostate cancer, androgenreceptor positive (AR)); MDA-MB-231 (breast cancer, estrogen receptor,progesterone receptor, and Her2 negative (triple negative)); A2058(melanoma); U-87 MG (glioblastoma); NCI-H460 (NSCLC); A549 (NSCLC); HPAC(pancreatic cancer, primary); PC3 (prostate cancer, AR⁻); BT474 (breastcancer, Her2 highly positive (Her2hi)); SKOV3 (ovarian cancer, Her2hi);786-O (renal cancer); UO-31 (renal cancer, MDR); NCI-H740 (SCLC); DMS53(SCLC); SK-BR3 (breast cancer, Her2hi); ZR-75 (breast cancer, estrogenreceptor positive); OVCAR3 (ovarian cancer); HL-60 (APL); OVCAR8/Adr(ovarian cancer, MDR); CEM-C1 (ALL); Nomo-1 (AML); RPMI-8226 (MM)); Raji(lymphoma); SW-480 (colorectal cancer, metastatic); SW-620 (colorectalcancer); and H226 (lung cancer). (Not all compounds were assayed againstall cell lines.)

The following compounds were used as reference or comparison cytotoxins:doxorubicin (Dox), Cytotoxin CBI (a DNA minor groove alkylating agent ofthe cyclopropa[c]benz[e]indol-4-one class), tubulysin D (Tub D, Table1), and the methyl ester of MMAF (“MMAF,” an auristatin-relatedcompound; see Sutherland et al., J. Biol. Chem. 2006, 281 (15),10540-10547).

FIGS. 11 a and 11 b show illustrative plots for ³H thymidineproliferation assays for compounds of this invention, against HL-60 and786-O cells, respectively, with Cytotoxin CBI and tubulysin D ascomparative compounds, with an incubation period of 48 h.

FIGS. 12 a and 12 b show illustrative plots for ATP luminescenceproliferation assays for a second set of compounds of this invention,against HL-60 and 786-O cells, respectively, with an incubation periodof 72 h. FIGS. 12 c and 12 d show the plots for ³H thymidineproliferation assays for the same set of compounds, again against HL-60and 786-O cells, respectively, with a 72 h incubation period. In eachinstance, doxorubicin was used as a comparative compound.

Table 2 provides the data for proliferation assays using the ³Hthymidine method, with an incubation period of 72 h.

TABLE 2 ³H Thymidine Proliferation Assays (Part 1) Cell Line and EC₅₀(nM) MDA- U-87 NCI- Compound HCT-15 Hep3B LNCaP MB-231 A2058 MG H460 Dox149 44 130 133 — 106 101 CBI 0.040 0.020 0.051 0.019 — 0.14 0.042 Tub D0.062 0.022 0.36 0.091 — 0.014 0.032 MMAF 1.3 0.11 1.7 0.29 — 0.12 0.3715 67 1.6 27 1.3 1.6 0.42 2.0 16 284 >100 >100 >100 >100 >100 >100 17392 >100 >100 84 >100 12 >100 19 1067 52 >100 — 0.13 >100 >100 24 704 3276 33 0.12 18 >100 25 280 305 930 236 409 271 306 26 31 18 >3000 15 255.2 18  26a >100 36 80 >100 >100 71 >100 28 223 24 64 >1000.96 >100 >100 29 0.18 0.041 0.54 0.13 0.40 0.064 0.21  29c 2.4 3.0 5.24.8 5.6 0.51 15 36 64 39 342 >100 60 15 52 51 101 4.2 41 8.7 0.40 0.757.2 54 0.44 0.30 16 1.6 0.64 0.11 0.31 562477 >100 >100 >100 >100 >100 >100 (Part 2) Cell Line and EC₅₀ (nM)Compound A549 HPAC PC3 BT474 SKOV3 786-O UO-31 Dox 128 126 276 424 163168 267 CBI 0.072 0.063 0.049 1.2 0.047 0.035 0.035 Tub D 0.014 0.0150.038 0.51 0.039 0.15 0.10 MMAF 0.21 0.29 0.38 1.7 0.24 1.4 7.3 (III-q)— — — — — 7.61 — 15 2.1 3.0 1.7 7.1 2.6 — 15 16 >100 >100 >100 >100 4419 >100 17 >100 >100 82 >100 91 32 >10019 >100 >100 >100 >100 >100 >100 >100 24 38 >100 63 >100 68 >100 >100 25270 206 319 804 180 1862 435 26 18 11 16 11 4.6 26 50 26a >100 >100 >100 7 47 >100 >100 28 >100 34 >100 >100 9.9 61 >100 290.44 0.069 0.21 0.57 0.029 1.2 0.13  29c 6.5 32 11 12 1.9 3.9 1.0 36 4483 66 >100 32 >100 >100 51 7.8 6.4 5.6 30 4.4 10 35 54 0.24 0.83 0.828.8 0.34 6.9 1.3 56 >100 >100 >100 >100 >100 29 >100 (Part 3) Cell Lineand EC₅₀ (nM) Compound H740 DMS53 SK-BR3 ZR-75 OVCAR3 HL-60 OVCAR8 Dox255 979 — 794 258 56 2044 CBI 0.098 1.5 — 1.5 0.071 0.027 0.16 Tub D 2.20.21 — 1.7 0.051 0.032 0.17 MMAF 13 10 — 42 0.16 0.25 25 (III-q) — — — —— 15.05 — 15 2.3 37 17 — >100 0.60 423 16 >100 >100 >100 >100 >100 8.91869 17 32 >100 >100 >100 >100 17 2970 19 3.8 >100 >100 34 53 37 69 2451 >100 >100 3.6 90 6.9 >100 25 347 515 1714 >3000 379 308 946 26 3.0 9932 >300 3.2 0.45 201  26a 3.2 >100 158 56 21 10 100 28 5.8 >100 >100 4.33.1 7.7 27 29 0.41 0.28 1.1 3.8 0.02 0.0099 0.70  29c 5.3 76 72 44 3.61.8 1.4 36 >100 >100 >100 >100 >100 107 >100 51 37 24 >100 1.3 2.5 4.220 54 10 2.0 1.1 2.2 3.9 0.53 2.1 56 >100 >100 >100 >100 >100 >100 >3000(Part 4) Cell Line and EC₅₀ (nM) Compound CEM-C1 Nomo-1 RPMI-8226 RajiBox 144 105 68 61 CBI 0.11 0.016 0.12 0.013 Tub D 0.018 0.042 0.0370.013 MMAF 0.17 0.27 0.24 0.12 15 0.93 2.3 3.3 0.47 16 11 1.8 22 28 1743 6.8 47 53 19 22 1.3 23 32 24 13 1.1 17 11 25 225 116 396 231 26 220.36 7.7 7.0  26a 10 2.0 22 20 28 10 0.20 8.7 13 29 0.019 0.0075 0.0340.024  29c 45 0.43 16 8.5 36 44 124 48 15 51 2.0 3.5 2.2 1.5 54 0.380.77 0.36 0.20 56 >100 >100 >100 >100

Table 3 provides the data for the ATP luminescence proliferation assays,with an incubation period of 48, 72, or 96 h, as noted.

TABLE 3 ATP Luminescence Proliferation Assays (Incubation Period asNoted)) (Part 1) Cell Line and EC₅₀ (nM) MDA- NCI- HCT-15 Hep3B MB-231A2058 H460 A549 PC3 Compound (48 h) (48 h) (48 h) (48 h) (48 h) (48 h)(48 h) Dox — 443 — — — 149   1082 CBI — 2.6 — — — 3.0 12 Tub D — 0.0012— — —  0.020 0.039 MMAF — 0.053 — — —  0.33 0.43 15  40 6.3   5.6   3.56.7 — >100 16 157 26 28 30 69 — >100 17 443 >100 65 30 69 — >100 19 — 18— — — — ~33 24 — 10 — — — — 13 25 — 246 — — — 111   155 26 113 100 120 93 — — >3000  26a — — — — — — — 28 — 17 — — — — 5.8 29 — 1.6 — — — 3.93.4  29c 200 75 52 32 — — 66 36 — 86 — — — 1.9 78 51 — 3.8 — — — — 16 54— 1.5 — — — 3.0 3.1 56 >3000  >100 >100  >100  >100 — >100 (Part 2) CellLine and EC₅₀ (nM) BT474 SKOV3 786-O UO-31 DMS53 SK-BR3 ZR-75 Compound(48 h) (48 h) (48 h) (48 h) (48 h) (48 h) (48 h) Dox — 674 360 908 — — —CBI — 3.0 1.6 4.2 — — — Tub D — 0.029 0.022 0.099 — — — MMAF — 0.19 0.712.6 — — — 15 3.5 4.2 — — 32 3.9 13 16 19 18 — — 50 6.6 11 17 23 58— >100 ~80 24 24 19 8.8 — — >100 — 3.9 20 24 4.6 — — 13 — 8.9 8.4 25 166— — 759 366 >3000 1003 26 19 — — 240 — 253 208  26a — — — — — — — 28 12— — 5.8 — 7.8 16 29 1.5 — — 2.7 5.3 3.6 15  29c 492 — — 26 — 278 136 36~120 — — 8.8 ~300 >300 >300 51 11 — — >100 — 13 19 54 4.8 — — — 7.9 6.29.6 56 >100 >100 — — >100 >100 >100  84a — — 6.12 — — — —  84b — — 4.10— — — — 109  — — 1.03 — — — — (Part 3) Cell Line and EC₅₀ (nM) OVCAR3HL-60 CEM-C1 Nomo-1 RPMI-8226 HCT-15 LNCaP Compound (48 h) (48 h) (48 h)(48 h) (48 h) (72 h) (72 h) Dox 632 — — — — 600 592 CBI 4.1 — — — — 3.43.3 Tub D 0.085 — — — — 0.014 0.093 MMAF 0.17 — — — — 0.075 0.98 15 >100— — — — — — 16 >100 — — — — — — 17 >100 — — — — — — 19 25 — — — — 245 —24 29 — — — — 188 — 25 >3000 224 — 83   — 366 — 26 — — 61 3.9 45 — 412 26a — — — — — — — 28 14 — — — — 132 — 29 >100 0.59 —  0.24 — 4.5 —  29c— — 20 2.9 70 — 255 36 >300 114 — 34   — 96 >300  36a 0.048 — — — — — 515.5 — — — — 93 — 54 >100 2.5 — 3.1 — 2.6 11 56 >100 — — — — — —  84a8.19 10-28 — — — 138 —  84b 4.91 16-27 — — — 22.8 — 109  0.019 — — — — —112  0.05-.09  — — — — — (Part 4) Cell Line and EC₅₀ (nM) MDA- U-87 NCI-MB-231 A2058 MG H460 A549 HPAC BT474 Compound (72 h) (72 h) (72 h) (72h) (72 h) (72 h) (72 h) Dox 518 101 324 92 — 215 388 CBI 3.1 0.20 3.30.64 — 6.0 5.5 Tub D 0.047 0.0064 0.0054 0.0076 — — 0.021 MMAF 0.330.039 0.052 0.13 — 0.067 0.024 15 — — 8.6 — — — — 16 — — >100 — — — — 17— — 22 — — — — 19 32 26 >100 43 — 0.33 — 24 13 14 18 23 — 1.1 — 25 202212 357 177 — 97 — 26 — — 94 90 — 279 —  26a — — — — — — — 28 16 20 1835 — 5.9 — 29 5.1 2.9 4.9 3.0 — 4.9 —  29c — — 136 6.0 — 4.6 — 36 9.6 4337 16 — 2.9 — 51 — 5.5 7.6 14 — 4.5 — 54 5.6 1.4 1.6 1.6 — 0.96 — 56 —— >100 — — — — (Part 5) Cell Line (EC₅₀, nM) SKOV3 786-O H740 DMS53SK-BR3 ZR-75 OVCAR3 Compound (72 h) (72 h) (72 h) (72 h) (72 h) (72 h)(72 h) Dox — — — 755 441 236 — CBI — — 2.2 3.8 3.1 2.2 — Tub D — — 0.0220.069 0.025 0.034 — MMAF — — — 4.5 0.16 0.25 — (III-q) — 103.6 — — — — —15 — 9.4 — — — — — 16 — 36 — — — — — 17 — 73 — — — — — 19 15 40 — 87 — —— 24 13 36 — >100 — — — 25 109  306 — — — — — 26 38 126 — 193 — — 49    26a — — — — — — — 28 11 29 — 58 — — — 29   1.3 2.8 — — — — —  29c 17 34— 17 — — 99    36 45 183 — — — — —  36a — 0.434 — — — — — 51 10 19 — 46— — — 54   1.3 2.6 — — — — — 56 — >100 — — — — — 109  — — — — — — 0.202112  — 0.67 — — — — 0.049 133  — 4.17 — — — — — 134  — 9.89 — — — — —135  — 12.94 — — — — — (Part 6) Cell Line and EC₅₀ (nM) HL-60 CEM-C1Nomo-1 RPMI-8226 Raji SW480 SW-620 Compound (72 h) (72 h) (72 h) (72 h)(72 h) (96 h) (96 h) Dox 147 161 338 345 301 215 145 CBI 0.76 0.55 0.374.1 2.1 0.92 0.33 Tub D 0.0096 0.0030 0.013 0.0051 0.0048 0.020 0.0058MMAF 0.12 0.11 0.14 0.068 0.084 0.17 0.057 (III-q) 14.27 — — — — — — 152.7 2.6 2.7 2.6 2.5 5.6 3.7 16 2.8 2.5 3.1 15 3.0 48 29 17 7.4 26 3.9 1527 92 65 19 7.7 11 5.2 9.6 9.2 52 14 24 6.4 8.7 4.0 8.4 7.9 42 10 25 —176 — 235 209 261 193 26 27 — — — 46 67 48  26a — — — — — — — 28 2.9 110.82 10 15 36 21 29 — 0.85 — 1.0 2.1 2.7 1.5  29c 8.6 — — — 36 41 8.4 36— 40 — 62 24 81 45 51 — 4.8 7.4 5.2 5.0 13 6.4 54 — 1.2 — 1.6 1.2 1.61.1 56 >100 >100 >100 5.7 24 >100 28

Additionally the following EC₅₀ values were measured for the followingcompounds against the H226 cell line, using the ATP assay and 72 hincubation period: compound 36a (0.307 nM); compound 109 (1.609 nM); andcompound 112 (0.67-1.16 nM). The following EC₅₀ values were measured forthe following compounds against the OVACAR8/Adr cell line, using the ATPassay and 48 h incubation period: compound 36a (17.05 nM); compound 84a(>300 nM); compound 84b (47.2 nM); compound 109 (24.9 nM); and compound112 (12 nM).

Example 15 Conjugate In Vitro Activity

FIG. 13 shows the activity of conjugates of this invention in ³Hthymidine proliferation assays, measured against 786-O renal cancercells, which are CD70 positive. The incubation period was 72 h. The EC₅₀values extracted from the curves of FIG. 13 are given in Table 4, alongdata from other experiments. Cell line LNCap is a prostate cancer cellline that expresses prostate specific membrane antigen (PSMA); H226 is alung cancer cell line that expresses mesothelin. The antibodies used forconjugation were 2A10, an anti-PSMA antibody (Huang et al., US2009/0297438); 2H5, an anti-CD70 antibody (Terrett et al., US2009/0028872); 1F4, an anti-CD70 antibody (Coccia et al., WO2008/074004); and 6A4, an anti-mesothelin antibody (Terrett et al., WO2009/045957). As controls, Compound J of Sufi et al., WO 2008/083312(“Cpd. J,” a DNA minor groove binding/alkylating agent) was used as aconjugation partner and diphtheria toxin (“DTX”) was used as anunconjugated non-specific control.

TABLE 4 In Vitro Activity of Conjugates Conjugate or Test Compound CellEC₅₀ Designation Description Line (nM) 2A10-Cpd. J Conjugate of 2A10 andCpd. J 786-O 116.7 DTX Unconjugated non-specific control 786-O 104.42H5-Cpd. J Conjugate of 2H5 and Cpd. J 786-O 0.08592 1F4-Cpd. JConjugate of 1F4 and Cpd. J 786-O 0.09951 2H5-(VI-a) Conjugate of 2H5and Compound (VI-a) 786-O 0.1151 to 0.0749 2A10-(VI-a) Conjugate of 2A10and Compound (VI-a) 786-O >>100 2H5-(VI-b) Conjugate of 2H5 and Compound(VI-b) 786-O 0.06554 2A10-(VI-b) Conjugate of 2A10 and Compound (VI-b)786-O >>100 2H5-(VI-n) Conjugate of 2H5 and Compound (VI-n) 786-O 0.43842H5-(VI-m) Conjugate of 2H5 and Compound (VI-m) 786-O 0.5899 2H5-(VI-q)Conjugate of 2H5 and Compound (VI-q) 786-O inactive 2H5-(VI-p) Conjugateof 2H5 and Compound (VI-p) 786-O inactive 2H5-(VI-t) Conjugate of 2H5and Compound (VI-t) 786-O 0.310 6A4-(VI-t) Conjugate of 6A4 and Compound(VI-t) H226 0.360 2A10-(VI-t) Conjugate of 2A10 and Compound (VI-t)LNCap 0.570

The data show that a CD70-specific antibody is needed for a conjugate tobe able to effectively deliver a cytotoxin to CD70-positive 786-O cells:conjugates of antibody 2A10, which is specific for a different antigen(PSMA), have low or no activity. Conversely, all the conjugates of ananti-CD70 antibody were active. The conjugates of Compounds (VI-a) and(VI-b) were comparable in activity to the Compound J conjugates, a wellknown conjugation partner and one of whose conjugates is undergoingclinical trials. It is noteworthy that the conjugates of Compounds(VI-a) and (VI-b) exhibit very little non-specific toxicity: compare theactivities of 2A10-(VI-a) and 2A10-(VI-b) against that of 2A10-CBI.

Example 16 Conjugate In Vivo Activity

CD70-positive human renal cancer 786-O cells (Cat. CRL-1932 originallyacquired from ATCC) were cultured in vitro per ATCC instructions. Thecells were harvested, and 2.5 million cells per 200 μL of DPBS/MATRIGEL™(1:1) were implanted subcutaneously in the flank region of CB17.SCIDmice. Tumors were measured weekly in 3 dimensions with a FowlerElectronic Digital Caliper (Model 62379-531; Fred V. Fowler Co., Newton,Mass., USA) and data were electronically recorded using StudyDirectorsoftware from Studylog Inc. (South San Francisco, Calif., USA). Animalswere checked daily for postural, grooming, and respiratory changes, aswell as lethargy. Animals were also weighed weekly and euthanized ifweight loss was ≧20%. When tumors reached an average size of 194 mm³,groups of 6 mice each were treated with a single intraperitoneal (IP)dose of a test conjugate (e.g., 2H5-(VI-b)) and an isotype control(2A10-(VI-b)) at 0.3 μmol/kg body weight. Tumor volumes (LWH/2) andweights of mice were recorded throughout the course of each study, whichwere allowed to proceed for approximately 2 months post initial dosing.An Excel spreadsheet macro was used to calculate the mean, SD, andmedian values of tumor sizes. Data were graphed using Prism softwareversion 4.0.

The xenograft study results are shown in FIG. 14, where legend labelshave the same meaning as in the previous Example and in FIG. 13. Thedata demonstrate the in vivo activity of conjugates of compounds of thisinvention against CD70⁺786-O cells. Both conjugates of compounds (VI-a)and (VI-b) with the anti-CD70 antibody 2H5 caused a reduction in meantumor size to less than half over the course of the study, while, whenthe vehicle control or a conjugate with the anti-PSMA antibody 2A10 wasadministered, tumor mean volume more than doubled.

Example 17 Scheme 13

Scheme 13 (FIG. 15) shows a method for making enantiomerically pure4-nitrotubuphenylalanines (4-NO₂Tup) 82a and 82b, which are useful formaking compounds of this invention.

Compound 80

Di-tert-butyl dicarbonate (90.5 mg, 0.42 mmol) was added to a mixture ofcompound 34 of Scheme 3 (0.1 g, 0.35 mmol) in 0.7 M aq. NaOH (1 mL). Thereaction mixture was stirred at RT for 3 h, and then acidified to pH 3with 0.5 N HCl. After the aqueous solution was extracted with EtOActhree times, the combined organic layers were dried, filtered, andconcentrated. The crude product was purified by flash chromatographyeluting from silica gel with a gradient of 0-20% methanol in DCM toafford 0.117 g of compound 80 as a white solid. MS: (+) m/z 253.1 (M+1without Boc).

(−)-Menthol esters 81a and 81b

DCC (87.8 mg, 0.43 mmol) was added to a solution of compound 80,(−)-menthol (66.6 mg, 0.43 mmol), and 4-(dimethylamino)-pyridine(“DMAP,” 10.4 mg, 0.085 mmol) in DCM (1.5 mL) at RT. After the reactionmixture was stirred at RT for 3 h, the precipitate was filtered off. Thefiltrate was then concentrated. The crude product was purified by flashchromatography eluting from silica gel with a gradient of 0-20% EtOAc inhexanes to afford 55.7 mg of ester 81a as a white solid, and 55.7 mg ofester 81b as a white solid. MS for ester 81a: (+) m/z 391.2 (M+1 withoutBoc); MS for ester 81b: (+) m/z 391.2 (M+1 without Boc).

4-NO₂Tup 82a and 82b

A solution of ester 81a in 6N HCl (40 mg, 0.082 mmol) was heated at 130°C. for 1.5 h. The reaction mixture was concentrated to afford 23.5 mg of4-NO₂Tup 82a as a white solid. ¹H NMR (D₂O, 400 MHz): δ 8.04 (d, 2H,J=8.4 Hz), 7.33 (d, 2H, J=8.4 Hz), 3.50 (m, 1H), 3.03 (dd, 1H, J=6.8,14.4 Hz), 2.89 (dd, 1H, J=7.6 Hz, 14.4 Hz), 2.45-2.39 (m, 1H), 1.92-1.84(m, 1H), 1.62-1.55 (m, 1H), and 0.98 (d, 3H, J=7.2 Hz); MS: (+) m/z253.1 (M+1). 4-NO₂Tup 82b was obtained by the same procedure on the samescale as a white solid (23.5 mg). ¹H NMR (D₂O, 400 MHz): δ 8.03 (d, 2H,J=8.4 Hz), 7.33 (d, 2H, J=8.4 Hz), 3.50 (m, 1H), 2.93 (dd, 2H, J=2.0,7.6 Hz), 2.54-2.48 (m, 1H), 1.86-1.78 (m, 1H), 1.60-1.53 (m, 1H), and0.98 (d, 3H, J=6.8 Hz); MS: (+) m/z 253.1 (M+1).

Example 18 Scheme 14

Scheme 14 (FIG. 16) depicts the conversion of 4-NO₂Tup 82a and 82b intocompounds of this invention.

Nitro Acid 83a

Compound 34a of Scheme 3 (10 mg, 0.019 mmol) was added to a 0.2 Msolution of pentafluorophenol (5.1 mg, 0.028 mmol) and N,N′-diisopropylcarbodiimide (“DIC,” 0.0032 mL, 0.021 mmol) in DCM (0.2mL) at 0° C. The reaction mixture was warmed to RT and stirred at suchtemperature overnight. The solvent was evaporated. EtOAc (1.8 mL) wasadded, and the crude product was filtered, with rinsing of the reactionvessel with EtOAc. The filtrate was concentrated under reduced pressure,and the crude pentafluorophenyl was used without further purification.DMF (0.2 mL) was added to the crude ester, followed by 4-NO₂Tup 82a(10.7 mg, 0.037 mmol), and DIEA (0.013 mL, 0.074 mmol). The reactionmixture was stirred at RT overnight, and the solvent was then evaporatedoff. The crude product was purified by flash chromatography eluting fromsilica gel with a gradient of 0-20% MeOH containing 1% NH₄OH in DCM toafford 12.9 mg of nitro acid 83a as a white solid. MS: (+) m/z 773.4(M+1).

Alternative Route Nitro Acid 83a

DIEA was added to a solution of compound 34a (10 mg, 0.019 mmol), andHATU (7.8 mg, 0.020 mmol) in DMF (0.3 mL), maintaining the pH at 8-9.The reaction mixture was stirred at RT for 5 min. Then DIEA and nitroamine 82a (5.4 mg, 0.019 mmol) in DMF (1 mL) were added, maintaining thepH at 8-9. After the reaction mixture was stirred at RT for 15 min, thecrude product was purified by preparative HPLC to afford 13.4 mg ofnitro acid 83a as a white solid.

Nitro acid 83b was prepared by the same alternative route, on the samescale and was obtained as a white solide (13.4 mg). MS: (+) m/z 773.4(M+1).

Amine 84a

A mixture of nitro acid 83a (7.5 mg, 0.0097 mmol) and palladium oncarbon (10%, 1.1 mg) in MeOH (0.37 mL) was stirred under a hydrogenatmosphere for 4 h. The catalyst was filtered off, and the filtrate wasconcentrated. The crude product was purified by preparative HPLC toafford 6.2 mg of amine 84a as a white solid. ¹H NMR (CD₃OD, 400 MHz): δ8.06 (s, 1H), 7.36 (d, 2H, J=8.4 Hz), 7.17 (d, 2H, J=8.4 Hz), 5.70 (dd,1H, J=2.8, 10.8 Hz), 4.71 (d, 1H, J=7.2 Hz), 4.44-4.35 (m, 2H), 3.74 (d,1H, J=9.6 Hz), 3.49-3.45 (m, 1H), 3.36-3.35 (m, 1H), 3.30-3.25 (m, 1H),3.13 (s, 3H), 3.14-3.04 (m, 1H), 2.93 (d, 2H, J=8.4 Hz), 2.74 (s, 3H),2.48-2.28 (m, 3H), 2.15 (s, 3H), 2.19-2.03 (m, 2H), 1.95-1.86 (m, 4H),1.80-1.71 (m, 2H), 1.71-1.57 (m, 3H), 1.24-1.13 (m, 1H), 1.16 (d, 3H,J=7.2 Hz), 1.04 (d, 3H, J=6.4 Hz), 1.02 (d, 3H, J=6.8 Hz), 0.94 (t, 3H,J=7.2 Hz), and 0.84 (d, 3H, J=6.8 Hz); MS: (+) m/z 743.4 (M+1).

Nitro acid 83b was hydrogenated to amine 84b using the same procedure,on an 8 mg scale. Amine 84b was obtained as a white solide (6.7 mg). ¹HNMR (CD₃OD, 400 MHz): δ 8.06 (s, 1H), 7.35 (d, 2H, J=8.4 Hz), 7.16 (d,2H, J=8.4 Hz), 5.70 (dd, 1H, J=2.8, 11.2 Hz), 4.72 (d, 1H, J=7.2 Hz),4.49-4.32 (m, 2H), 3.75 (d, 1H, J=10.0 Hz), 3.49-3.45 (m, 1H), 3.36-3.35(m, 1H), 3.33-3.31 (m, 1H), 3.12 (s, 3H), 3.12-3.04 (m, 1H), 2.91 (d,2H, J=7.6 Hz), 2.74 (s, 3H), 2.57-2.52 (m, 1H), 2.45-2.37 (m, 1H),2.33-2.28 (m, 1H), 2.15 (s, 3H), 2.19-2.13 (m, 1H), 2.03-1.88 (m, 5H),1.81-1.57 (m, 5H), 1.24-1.13 (m, 1H), 1.17 (d, 3H, J=6.8 Hz), 1.04 (d,3H, J=6.4 Hz), 1.02 (d, 3H, J=7.2 Hz), 0.94 (t, 3H, J=7.2 Hz), and 0.84(d, 3H, J=6.4 Hz); MS: (+) m/z 743.4 (M+1).

Compounds 84a and 84b are also depicted in this specification byformulae (III-r) and (III-s), respectively.

Example 19 Scheme 15

Scheme 15 (FIG. 17) depicts the synthesis of conjugation-ready compoundsof this invention having a single amino acid (citrulline) linker.

Compound 85

A mixture of compound 62 of Scheme 10 (0.22 g, 0.654 mmol),Fmoc-protected citrulline (0.39 g, 0.981 mmol), andN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (“EDCI,”0.188 g, 0.981 mmol) in DMF (4 mL) was stirred at RT overnight. Thereaction was quenched by addition of saturated NH₄Cl, and the aqueoussolution was extracted with EtOAc. The combined organic layers weredried, filtered, and concentrated. The crude product was purified byflash chromatography, eluting from silica gel with a gradient of 0-100%MeOH in DCM to afford 0.42 g of compound 85 as a white solid. MS: (+)m/z 716.4 (M+1).

Compound 86

Piperidine was added to a solution of compound 85 (0.248 g, 0.346 mmol)in DMF, maintaining the pH at 9-10. The reaction mixture was stirred atRT for 20 min, and then concentrated to afford 0.17 g of compound 86.MS: (+) m/z 494.4 (M+1).

Compound 87

LiOH (26.6 mg, 1.11 mmol) in water (3 mL) was added to a solution ofcompound 86 (0.17 g, 0.346 mmol) in THF (2 mL). After the reactionmixture was stirred at RT for 2 h, the solvent was partially removed.The aqueous solution was acidified to pH 2-3 with HCl and concentrated.The residue was re-dissolved in DMF (2 mL), and N-succinimidyl6-maleimidohexanoate (0.16 g, 0.519 mmol) and DIEA (0.091 mL, 0.519mmol) were added. After the reaction mixture was stirred at RT for 10min, the crude product was purified by preparative HPLC to afford 0.198g of compound 87 as a white solid. MS: (+) m/z 673.4 (M+1).

Compound 88

TFA (0.5 mL) was added to a solution of compound 87 (12.5 mg, 0.019mmol) in DCM (0.5 mL) at room temperature. The reaction mixture wasstirred at RT for 5 min, and then concentrated to afford 12.8 mg ofcompound 88 as a white solid. MS: (+) m/z 573.4 (M+1).

Compound 89

DIEA was added to a solution of compound 34a of Scheme 3 (5 mg, 0.0093mmol) and HATU (3.9 mg, 0.010 mmol) in DMF (0.3 mL), maintaining the pHat 8-9. The reaction mixture was stirred at RT for 5 min. Then DIEA andcompound 88 (12.8 mg, 0.019 mmol) in DMF (1 mL) were added, maintainingthe pH at 8-9. After the reaction mixture was stirred at RT for 15 min,the crude product was purified by preparative HPLC to afford 8.6 mg ofcompound 89 as a white solid. MS: (+) m/z 1093.8 (M+1). Compound 89 isalso depicted in this specification by formula (VI-m).

Compound 90

DIEA was added to a solution of compound 49 of Scheme 5 (5 mg, 0.0098mmol) and HATU (4.1 mg, 0.011 mmol) in DMF (0.3 mL), maintaining the pHat 8-9. The reaction mixture was stirred at RT for 5 min. Then DIEA andcompound 88 (13.5 mg, 0.0196 mmol) in DMF (1 mL) were added, maintainingthe pH 8-9. After the reaction mixture was stirred at RT for 15 min, thecrude product was purified by preparative HPLC to afford 8.9 mg ofcompound 90 as a white solid. MS: (+) m/z 1065.6 (M+1). Compound 90 isalso depicted in this specification by formula (VI-p).

Example 20 Scheme 16

Scheme 16 (FIG. 18) depicts the preparation of conjugation-readycompounds of this invention, having a dipeptide (citrulline-valine)linker.

Compound 91

DIEA was added to a solution of Fmoc-protected valine (62.3 mg, 0.184mmol) and HATU (63.6 mg, 0.167 mmol) in DMF (0.5 mL), maintaining the pHat 8-9. The reaction mixture was stirred at RT for 5 min. Then DIEA andcompound 86 of Scheme 15 (82.5 mg, 0.167 mmol) in DMF (1 mL) were added,maintaining the pH at 8-9. After the reaction mixture was stirred at RTfor 15 min, the reaction was quenched by addition of 0.05% aq. TFA. Theaqueous solution was extracted with EtOAcef and the combined organiclayers were dried, filtered and concentrated. The crude product waspurified by flash chromatography eluting from silica gel with a gradientof 0-20% MeOH in DCM to afford 0.13 g of compound 91 as a white solid.MS: (+) m/z 815.5 (M+1).

Compound 92

Piperidine was added to a solution of compound 91 (0.144 g, 0.177 mmol)in DMF, maintaining the pH at 9-10. The reaction mixture was stirred atRT for 20 min and then concentrated. The residue was dissolved in THF(2.5 mL) and LiOH (16.3 mg, 0.681 mmol) in water (1.3 mL) was added.After the reaction mixture was stirred at RT for 2 h, the solvent waspartially removed. The aqueous solution was acidified to pH 2-3 with HCland then concentrated. The residue was re-dissolved in DMF (2.5 mL), andthen N-succinimidyl 6-maleimidohexanoate (0.105 g, 0.341 mmol) and DIEA(0.060 mL, 0.341 mmol) were added. After the reaction mixture wasstirred at RT for 10 min, the crude product was purified by preparativeHPLC to afford 0.116 g of compound 92 as a white solid. MS: (+) m/z772.5 (M+1).

Compound 93

TFA (0.6 mL) was added to a solution of compound 92 (14.4 mg, 0.019mmol) in DCM (1 mL) at RT. The reaction mixture was stirred at RT for 5min and then concentrated to afford 14.7 mg of compound 93 as a whitesolid. ¹H NMR (CD₃OD, 400 MHz): δ 7.58 (dd, 2H, J=1.6, 8.4 Hz), 7.21(dd, 2H, J=2.8, 8.8 Hz), 6.79 (s, 2H), 4.48 (m, 1H), 4.13 (d, 1H, J=7.6Hz), 3.57-3.46 (m, 3H), 3.33-3.32 (m, 1H), 3.22-3.09 (m, 2H), 2.91-2.80(m, 1H), 2.27 (t, 2H, J=7.2 Hz), 2.09-1.85 (m, 3H), 1.81-1.54 (m, 8H),1.35-1.29 (m, 3H), 1.19 (d, 1.5 H, J=6.8 Hz), 1.18 (d, 1.5 H, J=7.2 Hz),0.98 (d, 3H, J=2.4 Hz), 0.96 (d, 3H, J=2.8 Hz); MS: (+) m/z 672.4 (M+1).

Compound 94

DIEA was added to a solution of compound 34a of Scheme 3 (11 mg, 0.0204mmol) and HATU (7.8 mg, 0.0204 mmol) in DMF (0.3 mL), maintaining the pHat 8-9. The reaction mixture was stirred at RT for 5 min. Then DIEA andcompound 93 (14.7 mg, 0.019 mmol) in DMF (1 mL) were added, maintainingthe pH at 8-9. After the reaction mixture was stirred at RT for 15 min,the crude product was purified by preparative HPLC to afford 18.9 mg ofcompound 94 as a white solid. MS: (+) m/z 1192.6 (M+1). Compound 94 isalso depicted in this specification by formula (VI-n).

Acetate of Compound 27

Acetic anhydride (0.248 mL) was added to a solution of compound 27 ofScheme 2 (Peltier et al., 2006; 0.13 g, 0.218 mmol) in pyridine (2.6 mL)at 0° C. The reaction mixture was then stirred at RT overnight. Afterthe reaction mixture was cooled at 0° C., a solution of water and1,4-dioxane (12 mL, v/v 1:1) was added. The reaction mixture was stirredat RT overnight and then concentrated. The crude product was purified byflash chromatography eluting from silica gel with a gradient of 10-20%MeOH in DCM to afford 0.114 g of the acetate of compound 27 as a whitesolid. MS: (+) m/z 639.4 (M+1).

Compound 95

DIEA was added to a solution of the acetate of compound 27 (3.8 mg,0.0059 mmol), and HATU (2.5 mg, 0.0065 mmol) in DMF (0.3 mL),maintaining the pH at 8-9. The reaction mixture was stirred at RT for 5min. Then DIEA and compound 93 (5.6 mg, 0.0071 mmol) in DMF (1 mL) wereadded, maintaining the pH at 8-9. After the reaction mixture was stirredat RT for 15 min, the crude product was purified by preparative HPLC toafford 6.5 mg of compound 95 as a white solid. MS: (+) m/z 1292.7 (M+1).Compound 95 is also depicted in this specification by formula (VI-o).

Example 21 Scheme 17

This example describes the synthesis of acid 108, an intermediate usefulfor the preparation of compounds of this invention, with reference toScheme 17 (FIG. 19).

Methyl Ester 100

HCl in dioxane (8.3 ml, 4M, 33.2 mmol) was added to a solution ofcompound 9 of Scheme 1 (8 g, 22.1 mmol) in MeOH (10 mL). The reactionmixture was stirred at RT. After 20 min, the solution was evaporatedunder vacuum to give methyl ester 100 as an oil (6.5 g), which was usedfor the next reaction step without further purification.

Propyl Amine 101

Propanal (700 μL, 7.36 mmol) and NaBH(OAc)₃ (2.8 g, 13.2 mmol) wereadded to a solution of methyl ester 100 (1.96 g, 6.6 mmol) in DCM (10mL). The reaction mixture was stirred at 5° C. After 1 h the mixture wastaken up in EtOAc and washed with 7% K₂CO₃ solution twice and thenbrine. The EtOAc layer was dried over anhydrous Na₂SO₄ and thenevaporated under vacuum to yield a residue which was passed through acolumn (MeOH:DCM. 0-10%) to give propyl amine 101 (1.12 g, 60%) as anoil. ¹H NMR (400 MHz, CDCl₃) δ 8.17 (s, 1H), 5.43 (t, J=4.6 Hz, 1H),3.93 (s, 3H), 3.07-2.87 (m, 2H), 2.82-2.70 (m, 1H), 2.54 (s, 1H),2.45-2.26 (m, 2H), 2.16-2.02 (m, 1H), 1.73 (m, 2H), 1.05-0.94 (m, 9H).MS m/z C₁₄H₂₅N₂O₃S (M+1)⁺ calculated 301.2. found 301.

Compound 102

(Benzotriazol-1-yloxy)tripyrrolidinophosphonim hexafluorophosphate(“PyBop,” 1.28 g, 2.47 mmol), HOBt (0.33 g, 2.47 mmol), Boc-protectedisoleucine (430 μL, 2.47 mmol) were added to a solution of propyl amine101 (570 mg, 1.9 mmol) in DCM (5 mL). The reaction mixture was stirredat RT. After 20 min EtOAc (200 mL) was added and the organic layer waswashed with 10% citric acid (twice), sat. NaHCO₃, and brine. The EtOAclayer was dried over anhydrous Na₂SO₄ and then evaporated under vacuumto yield a residue which was passed through a column to give compound102 (0.55 g) as an oil. MS m/z C₂₅H₄₄N₃O₆S (M+1)⁺ calculated 514.3.found 514.3.

Azido Ester 104

Acid chloride 103 (2 mmol, Lundquist et al. 2001; see also above in thepreparation of azido ester 43 of Scheme 5) in DCM (3 mL) was added to asolution of compound 102 (0.55 g, 1.1 mmol) in DCM (10 mL) and DIEA (871μL, 5 mmol). The reaction mixture was stirred at 5° C. After stirringfor 10 min the mixture was evaporated under vacuum to give a residuewhich was passed through a column to give azido ester 104 (300 mg) as anoil. MS m/z C₃₁H₅₃N₆O₂S (M+1)⁺ calculated 653.4. found 653.

Compound 106

A solution of pentafluorophenyl ester 105 (2.1 mmol, Peltier et al.2006) in 1 mL of EtOAc was added to a solution of azido ester 104 (300mg, 0.46 mmol) and Pd/C (10%, 50 mg) in EtOAc (5 mL). The reaction flaskwas filled with H₂ using a balloon and was stirred overnight at RT.After stirring overnight the reaction mixture was filtered, concentratedunder vacuum and then passed through a column (MeOH:DCM, 0-10%) to givecompound 106 (170 mg) as an oil. MS m/z C₃₈H₆₆N₅O₈S (M+1)⁺ calculated752.5. found 752.5.

Compound 107

NaOH (120 uL, 1.2 mmol, 10M) was added to a solution of compound 106(170 mg, 0.22 mmol) in MeOH (10 mL) at RT. After stirring for 2 hr thereaction mixture was acidified to pH 2 with concentrated HCl. Thereaction mixture was then evaporated under vacuum and passed through areverse phase column (ACN:H₂O, 0-100% with 0.1% TFA). Afterlyophilization, compound 107 (63 mg) was obtained as a white powder.HPLC profile indicated it was a mixture of rotamers. MS m/z C₂₆H₄₅N₄O₅S(M+1)⁺ calculated 525.3. found 525.

Acid 108

Acetic anhydride (60 uL, 0.64 mmol) was added to a solution of compound107 (63 mg, 0.12 mmol) in pyridine (1 mL) at 5° C. The temperature wasraised to RT gradually. After allowing reaction to proceed overnight,water (100 uL) was added. After another 5 hr, the volatile organics wereremoved under vacuum to give a residue which was passed through areverse phase column (ACN:H₂O, 0-100% with 0.1% TFA) to give acid 108(42 mg) as an oil. ¹H NMR (400 MHz, CD₃OD) δ 8.35 (s, 1H), 5.71 (dd,J=11.4, 1.4 Hz, 1H), 4.63 (d, J=9.1 Hz, 1H), 3.97 (t, J=16.4 Hz, 1H),3.65-3.42 (m, 2H), 3.21-3.05 (m, 2H), 2.87 (s, 3H), 2.34-2.14 (m, 4H),2.13 (s, 3H), 2.03-1.46 (m, 10H), 1.29-1.06 (m, 1H), 1.04-0.85 (m, 15H).MS m/z C₂₈H₄₂N₄O₅S (M+1)⁺ calculated 567.3. found 567.

Example 22 Schemes 18 and 19

Scheme 18 (FIGS. 20 a and 20 b) shows the synthesis of compounds of thisinvention using as acid 108, prepared in the preceding example.

General Procedure for HATU-Mediated Coupling

HATU (1.2× excess) and DIEA (4× excess) were added into a solution ofacid 108 in DMF at 5° C. After stirring the reaction mixture for 10 min,the corresponding amine was added. The reaction mixture was stirred foranother 10 min before diluting it with DMSO and 0.1% TFA solution. Theresulting mixture was passed through a reverse phase column (ACN:H₂O,0-100% with 0.1% TFA). The collected fractions were analyzed and thedesired fractions were lyophilized to give the corresponding product.

Compound 109

Obtained from the coupling of acid 108 and phenylalanine methyl ester.MS m/z C₃₈H₅₈N₅O₇S (M+1)⁺ calculated 728.4. found 728.4. Compound 109 isalso depicted hereinabove as structure (III-x).

Compound 111

Obtained from the coupling of acid 108 and compound 110 (preparationdescribed below). MS m/z C₄₂H₆₃N₆O₉S (M+1)⁺ calculated 827.4. found827.5.

Compound 112

To a solution of compound 111 (5 mg, 6 μmol) in 2 mL of MeOH was addedPd/C (10%, 10 mg). The reaction flask was filled with H₂ using a balloonand was stirred for 2 h at RT. The reaction mixture was then filtered,concentrated under vacuum and passed through a reverse phase column(ACN:H₂O, 0-100% with 0.1% TFA) to give compound 112 (2.1 mg) as a whitepower. MS m/z C₄₂H₆₂N₆O₂S (M+1)⁺ calculated 799.5. found 799.5. Compound112 is also depicted hereinabove as structure (III-y).

Compound 114

Obtained from the coupling of acid 108 and compound 113 (preparationdescribed below). MS m/z C₄₁H₆₄N₅O₇S (M+1)⁺ calculated 770.4. found 770.

Compound 116

Obtained from the coupling of acid 108 and compound 115 (preparationdescribed below). MS m/z C₆₁H₉₅N₁₁O₁₃S (M+2)⁺ calculated 610.9. found611. Compound 116 is also depicted hereinabove as structure (VI-t).

Compound 117

Obtained from the coupling of acid 108 and alpha-N-acetyl lysine methylester. MS m/z C₃₇H₆₃N₆O₈S (M+1)⁺ calculated 751.4. found 751.5.

Compound 110

Alkene 59 of Scheme 8 (as ethyl ester instead of methyl ester, 1 g, 2.6mmol) was dissolved in DCM (10 mL) containing 5% TFA and the reactionmixture was stirred at 5° C. After 40 min the mixture was dried undervacuum to give compound 110 (0.3 g, 100%) as a semi-solid. ¹H NMR (400MHz, CD₃OD) δ 8.22-8.17 (m, 2H), 7.50 (dd, J=9.0, 2.2 Hz, 2H), 6.58 (d,J=10.0 Hz, 1H), 4.45 (td, J=9.8, 5.3 Hz, 1H), 4.19 (q, J=7.1 Hz, 2H),3.35-3.28 (m, 1H), 3.06 (dd, J=13.2, 9.6 Hz, 1H), 1.55 (d, J=0.9 Hz,3H), 1.27 (t, J=7.1 Hz, 3H).

Compound 113

HCl (2.5 mL, 10 mmol, 4 M) was added to a solution of compound 118 (2 g,5.5 mmol, Peltier et al. 2006) in MeOH (10 mL) and the reaction mixturewas stirred at RT. After 20 min the reaction mixture was dried undervacuum to give compound 113 (2 g, 100%) as a semi-solid. Crude productwas used for the next step reaction without further purification. MS m/zC₁₃H₁₉NO₂ (M+1)⁺ calculated 222.1. found 222.

Scheme 19 (FIG. 21) shows the synthesis of compound 115, used in thesynthesis of compound 116, above.

Compound 120

DIEA (697 μL, 12 mmol) and valine t-butyl ester 543 (627 mg, 3 mmol)were added into a 10 mL DCM solution of 6-maleimidohexanoic acid(“6-MHA,” 622 mg, 3 mmol) and HATU (1.14 g, 3 mmol). After 20 min EtOAc(200 mL) was added. The organic phase was washed with 10% citric acid,sat. NaHCO₃ solution, and brine. It was then dried with anhydrous Na₂SO₄and the solvent was removed by evaporation. The resulting residue waspassed through a column (Hexane:EtOAc, 0-80%) to give compound 120 (900mg) as an oil. ¹H NMR (400 MHz, CDCl₃) δ 6.66 (s, 2H), 5.94 (d, J=8.5Hz, 1H), 4.44 (dd, J=8.7, 4.5 Hz, 1H), 3.49 (t, J=7.2 Hz, 2H), 2.31-2.06(m, 3H), 1.73-1.54 (m, 4H), 1.45 (s, 9H), 1.37-1.25 (m, 2H). MS m/zC₁₉H₃₁N₂O₅ (M+1)⁺ calculated 367.2. found 367.

Compound 121

Compound 120 (1 g, 2.73 mmol) was dissolved in 20 mL DCM with 3 mL TFAat RT. After 1 h the mixture was dried by evaporation to give compound121 (1 g) as an oil, which was used without further purification. MS m/zC₁₅H₂₃N₂O₅ (M+1)⁺ calculated 311.2. found 311.

Compound 123

DIEA (920 μL, 5.28 mmol) and compound 122 (500 mg, 1.32 mmol; see Scheme22 and Example 25 hereinbelow) were added a 10 mL DMF solution ofFmoc-protected citrulline (524 mg, 1.32 mmol) and HATU (601 mg, 1.58mmol). After 20 min, 200 mL EtOAc was added. The organic phase waswashed with 10% citric acid, sat. NaHCO₃ solution, and brine. It wasthen dried with anhydrous Na₂SO₄ and the EtOAc was evaporated. Theresulting residue was passed through a column (MeOH:DCM; 0-10%) to givea solid. This solid was dissolved in DMF (5 mL) with 5% piperidine.After 1 h the solution was evaporated and the residue was passed througha reverse phase column (ACN:H₂O; 0-100% with 0.1% TFA) to give compound123 (212 mg). MS m/z C₂₂H₄₆N₅O₆ (M+1)⁺ calculated 536.3. found 536.4.

Compound 124

DIEA (404 μL, 2.4 mmol) and compound 560 (321 mg, 0.6 mmol) were addedinto a 5 mL DMF solution of compound 550 (180 mg, 0.58 mmol) and HATU(220 mg, 0.58 mmol). After 20 min 200 mL EtOAc was added. The organicphase was washed with 10% citric acid, sat. NaHCO₃ solution, and brine.It was then dried with anhydrous Na₂SO₄ and the EtOAc evaporated. Theresulting residue was passed through a column (MeOH:DCM; 0-20%) to givecompound 124 (240 mg) as an oil. MS m/z C₄₂H₆₆N₇O₁₀ (M+1)⁺ calculated828.5. found 828.5.

Compound 115

Compound 124 (240 mg, 0.29 mmol) was dissolved in a 5 mL solution of TFAand DCM (1:1). After 3 h the mixture was dried by evaporation and theresulting compound 115 was used without further purification. From NMR,a mixture of two (5:1) isomers was obtained. The major isomer isreported: ¹H NMR (400 MHz, CD₃OD) δ 8.27 (d, J=7.5 Hz, 1H), 7.58 (dd,J=8.5, 1.9 Hz, 2H), 7.21 (d, J=8.5 Hz, 2H), 6.79 (s, 2H), 4.48 (dd,J=13.3, 8.1 Hz, 1H), 4.14 (dd, J=7.5, 4.9 Hz, 1H), 3.62-3.38 (m, 3H),3.25-2.97 (m, 3H), 2.96-2.78 (m, 2H), 2.70-2.40 (m, 1H), 2.32-2.21 (m,2H), 2.11-1.92 (m, 2H), 1.94-1.83 (m, 1H), 1.82-1.70 (m, 1H), 1.70-1.49(m, 7H), 1.19 (d, J=7.0 Hz, 3H), 0.97 (dd, J=6.8, 2.8 Hz, 6H). MS m/zC₃₃H₅₀N₇O₈ (M+1)⁺ calculated 672.4. found 672.

Example 23 Scheme 20

Scheme 20 (FIG. 22) shows the synthesis of compound 131, an intermediateused for making compounds of this invention.

Compound 125

Compound 9 of Scheme 1 (3 g, 8.29 mmol) was dissolved in THF (20 mL) anddimethylsufate (1.2 mL, 12.4 mmol). To this solution was added NaH (552mg, 13.8 mmol) at 5° C. in portions over 1.5 h. The reaction mixture wasthen poured into sat. NH₄Cl solution. EtOAc was added to the reactionmixture and the organic phase was washed with brine and dried andevaporated under vacuum to give a residue. The resulting residue waspassed through a column (Hexane:EtOAc, 0-100%) to give compound 125 (1.2g) as an oil. ¹H NMR (400 MHz, CDCl₃) δ 8.12 (s, 1H), 4.95 (dd, J=10.0,3.3 Hz, 1H), 3.89 (s, 3H), 3.50 (s, 3H), 3.45-3.40 (m, 2H), 1.93-1.79(m, 2H), 1.74-1.65 (m, 1H), 1.20 (s, 9H), 0.84 (d, J=6.8 Hz, 3H), 0.80(d, J=6.8 Hz, 3H). MS m/z C₁₆H₂₉N₂O₄S₂ (M+1)⁺ calculated 377.1. found377.2.

Compound 126

HCl in dioxane (1 ml, 4 mmol) was added to a solution of compound 125(0.7 g, 1.86 mmol) in MeOH (10 mL). The reaction mixture was stirred atRT. After 20 min, the volatiles were evaporated under vacuum to givecompound 126 (0.8 g) as an oil which was used for the next reaction stepwithout further purification. MS m/z C₁₂H₂₁N₂O₃S (M+1)⁺ calculated273.1. found 273.

Compound 127

To a solution of compound 126 (616 mg, 2 mmol) in DCM (10 mL) and DIEA(1.8 mL, 10 mmol) at 5° C., was added compound 103 (Scheme 17, 6 mmol)in 5 mL of DCM. The reaction mixture was stirred for 3 h at RT. After 3h the reaction mixture was poured into sat. NaHCO₃ solution and EtOAc.The organic phase was washed with brine, dried, and evaporated. Theresulting residue was passed through a column (Hexane:EtOAc, 0-50%) togive compound 127 (594 mg, 72%) as a semi oil. ¹H NMR (400 MHz, CDCl₃) δ8.18 (s, 1H), 6.47 (d, J=9.9 Hz, 1H), 4.60-4.52 (m, 1H), 4.23-4.13 (m,1H), 3.96-3.95 (m, 1H), 3.94 (s, 3H), 3.44 (s, 3H), 2.21-2.08 (m, 1H),1.94-1.84 (m, 2H), 1.84-1.71 (m, 1H), 1.52-1.38 (m, 1H), 1.35-1.20 (m,1H), 1.07 (d, J=6.9 Hz, 3H), 0.95-0.85 (m, 9H). ¹³C NMR (101 MHz, CDCl₃)δ 176.00, 168.63, 161.92, 146.90, 128.24, 78.81, 70.34, 58.97, 52.72,50.76, 40.48, 38.62, 32.24, 24.32, 19.13, 18.13, 16.25, 11.82. MS m/zC₁₈H₃₀N₅O₄S (M+1)⁺ calculated 412.2. found 412.3.

Compound 129

Potassium hexamethyldisilazide (“KHMDS,” 0.19 mmol, 0.375 mL of toluenesolution) was added into a THF (0.5 mL) solution of compound 127 (50 mg,0.12 mmol) at −43° C. After 20 min compound 128 (0.36 mmol, 137 uL, Abeet al. 1997) was added. After 2 hr, 100 μL MeOH was added and thereaction mixture was poured into sat. NH₄Cl solution. EtOAc was thenadded. After separation of the layers, the organic phase was washed withbrine, dried with anhydrous Na₂SO₄ and the solvent removed byevaporation. The resulting residue was passed through a column(Hexane:EtOAc, 0-50%) to give compound 129 (51 mg) as a semi solid. ¹HNMR (400 MHz, CDCl₃) δ 8.16 (s, 1H), 5.70 (s, 1H), 5.43 (d, J=12.4 Hz,1H), 5.32 (d, J=12.3 Hz, 1H), 4.39 (d, J=10.6 Hz, 1H), 3.92 (d, J=11.7Hz, 3H), 3.53-3.44 (m, 1H), 3.37 (d, J=10.5 Hz, 3H), 2.41 (d, J=7.2 Hz,2H), 2.37-2.11 (m, 4H), 1.92-1.68 (m, 2H), 1.37-1.21 (m, 1H), 1.12-0.85(m, 18H). ¹³C NMR (101 MHz, CDCl₃) δ 175.24, 172.79, 171.21, 161.90,147.09, 128.25, 78.44, 68.69, 63.35, 58.71, 52.48, 43.23, 38.63, 34.91,31.01, 25.73, 25.25, 22.58, 22.56, 22.48, 20.45, 19.62, 16.14, 10.65. MSm/z C₂₄H₄₀N₅O₆S (M+1)⁺ calculated 526.3. found 424.3 (breaking of theN,O acetal).

Compound 130

Compounds 129 (200 mg, 0.38 mmol) and 105 (Peltier et al. 2006; 4 mmol)were mixed in 5 mL EtOAc with Pd/C (150 mg, 10%) at rt. The reactionflask was evacuated and refilled with H₂ using a balloon. After stirringovernight at RT, the mixture was filtered and the solvent evaporated.After column chromatography (SiO₂, MeOH:DCM, 0-10%), compound 130 (97mg) was obtained as a solid. MS m/z C₃₁H₅₃N₄O₇S (M+1)⁺ calculated 625.4.found 625.5.

Compound 131

Tribuytltin hydroxide (181 mg, 0.59 mmol) was added to a solution ofcompound 130 (97 mg, 0.16 mmol) in 10 mL 1,2-dichloroethane. After 22 hat 67° C., the mixture was evaporated and passed through a reverse phasecolumn (ACN:(20 mM NH₄(HCO₃) buffer, pH 7), 5-100%) to give compound 131(34 mg) as a solid. MS m/z C₃₀H₅₁N₄O₇S (M+1)⁺ calculated 611.3. found510 (break at the N,O acetal).

Example 24 Scheme 21

Scheme 21 (FIG. 23) shows the synthesis of compounds of this inventionusing compound 131 as a precursor.

Compound 132

Compound 60 (Scheme 8, 200 mg, 0.57 mmol) was dissolved in 2 mL DCM with20% TFA at rt. After 1 h the volatiles were evaporated to give compound132 (200 mg) as a yellow solid, which was used without furtherpurification.

Compound 133

DIEA (43 μL, 0.2 mmol) was added to a DMF (1 mL) solution of compound131 (30 mg, 0.049 mmol) and HATU (22.3 mg, 0.059 mmol) at −43° C. After10 min compound 132 (15 mg, 0.06 mmol) was added. The mixture was thenraised to RT. The final mixture was passed through a reverse phasecolumn (ACN:(20 mM NH₄(HCO₃) buffer, pH 7), 5-100%) to give compound 133(20 mg) as a white powder. MS m/z C₄₄H₇₃N₆O₈S (M+1)⁺ calculated 843.5.found 843.5. Compound 133 is also depicted hereinabove as structure(III-u).

Compound 134

Compound 133 (2 mg, 2.4 μmol) was dissolved in 0.5 mL methanol and thepH of the solution was adjusted to 1 with 1M HCl. After stirringovernight, the volatiles were evaporated and the residue was passedthrough a reverse phase column (ACN:(20 mM NH₄(HCO₃) buffer, pH 7),5-100%) to give compound 134 (0.7 mg) as a white powder. MS m/zC₄₀H₆₅N₆O₇S (M+1)⁺ calculated 773.5. found 773.5. Compound 134 is alsodepicted hereinabove as structure (III-v).

Compound 135

Compound 133 (2 mg, 2.4 μmol) was dissolved in 0.5 mL n-propanol and thepH of the solution was adjusted to 1 withlM HCl. After stirringovernight, the mixture was evaporated and the residue was passed througha reverse phase column (ACN:(20 mM NH₄(HCO₃) buffer, pH 7), 5-100%) togive compound 135 (0.4 mg) as a white powder. MS m/z C₄₂H₆₉N₆O₇S (M+1)⁺calculated 802.5. found 801.5. Compound 135 is also depicted hereinaboveas structure (III-w).

Example 25 Scheme 22

Scheme 22 (FIG. 24) shows a method for making compound 142, useful as anintermediate for making compounds of this invention.

Compound 136

NaOH (800 uL, 10M, 8 mmol) was added to a 20 mL solution of THF and MeOH(1:1) with compound 59 of Scheme 8 (1.65 g, 4.37 mmol). After stirringovernight the pH of the solution was adjusted to 1 with 3N HCl at 5° C.After evaporation of the solvents, 200 mL EtOAc was added. Afterseparation, the organic phase was washed with brine, dried withanhydrous Na₂SO₄ and the EtOAc evaporated. The residue was passedthrough a column (MeOH:DCM; 0-10%) to give compound 136 (1.2 g) as anoil. ¹H NMR (400 MHz, CDCl₃) δ 8.21-8.12 (m, 2H), 7.41-7.32 (m, 2H),6.62 (d, J=8.8 Hz, 1H), 4.82-4.57 (m, 2H), 3.15-3.02 (m, 1H), 2.90 (dd,J=13.3, 7.2 Hz, 1H), 1.71 (d, J=1.2 Hz, 3H), 1.41 (s, 9H).

Compound 137

DMF-di-t-Butylacetal (1 mL, 4 mmol) was added to a 6 mL toluene solutionof compound 136 (128 mg, 0.36 mmol) at 133° C. After 10 min the reactionmixture was cooled and the solvent evaporated. The residue was passedthrough a column (Hexane:EtOAc; 0-30%) to give compound 137 (133 mg) asan oil. ¹H NMR (400 MHz, CDCl₃) δ 8.19-8.10 (m, 2H), 7.39-7.30 (m, 2H),6.39 (dd, J=9.1, 1.5 Hz, 1H), 4.63 (d, J=39.1 Hz, 2H), 3.03 (dd, J=13.2,6.2 Hz, 1H), 2.90 (dd, J=13.3, 7.0 Hz, 1H), 1.67 (d, J=1.5 Hz, 3H), 1.47(s, 9H), 1.39 (s, 9H).

Compound 122

Compound 137 (540 mg, 1.22 mmol), Pd/C (136 mg, 10%) and 3N HCl (0.3 mL)were added to a mixture of DCM and MeOH (30 mL:5 mL). The flask wasfilled with H₂ using a balloon. After stirring overnight at RT, themixture was filtered and concentrated to give compound 122 (550 mg) as asemi-solid. MS m/z C₂₁H₃₅N₂O₄ (M+1)⁺ calculated 379.3. found 223 (lossof Boc).

Compound 138

Compound 136 (100 mg, 0.28 mmol) and Pd/C (20 mg, 10%) were mixed in a 5mL mixture of MeOH and DCM (1:1 v:v) under a hydrogen balloon at RT.After stirring overnight the mixture was filtered and the solventsevaporated under vacuum to give compound 138 (95 mg) as oil, which wasused for next reaction step without further purification. MS m/zC₁₇H₂₇N₂O₄ (M+1)⁺ calculated 323.2. found 223.

Compound 139

Compound 138 (10 mg, 0.03 mmol), tert-butyldimethylsilyl chloride(“TBDMSCl,” 4.5 mg, 0.03 mmol) and imidazole (4 mg, 0.06 mmol) weremixed in 1 mL DMF at RT. Fmoc-protected citrulline (24 mg, 0.06 mmol),N,N′-disuccinimidyl oxalate (“DSO,” 8 mg, 0.06 mmol) and DIEA (20 uL,0.12 mmol) were mixed in another 1 mL of DMF at RT. After 1 h the twosolutions were mixed and the mixture was kept at RT. After stirringovernight, EtOAc was added and the solution was washed with 10% aq.citric acid and brine. The organic phase was then dried with anhydrousNa₂SO₄ and evaporated under vacuum. The resulting residue was passedthrough a column (MeOH:DCM, 0-10%) to give compound 139 (7 mg) as anoil. MS m/z C₃₈H₄₈N₅O₈ (M+1)⁺ calculated 702. found 702.

Compound 141

Compound 139 (10 mg, 0.014 mmol) was dissolved in 1 mL DMF with 5%piperidine. After 20 min the solvent was evaporated under vacuum and theresidue was mixed with N-succinimidyl-4-maleimidobutyrate 140 (5.6 mg,0.028 mmol) and DIEA (5 uL, 0.028 mmol) in 1 mL DMF. After 10 min thesolvents were removed from the reaction mixture under vacuum and passedthrough a column (MeOH:DCM, 0-20%) to give compound 141 (6 mg) as asolid. MS m/z C₃₁H₄₅N₆O₉ (M+1)⁺ calculated 645. found 645.

Compound 142

Compound 141 (6 mg, 0.01 mmol) was dissolved in 1 mL DCM with 10% TFA.After 10 min the solvent was evaporated under vacuum to give compound142 (6 mg), used for next reaction step without further purification.

Example 26 Scheme 23

Scheme 23 (FIG. 25) shows the elaboration of compound 142, made perScheme 22, into a compound of this invention.

Compound 145

HCl (30 μmmol) in 150 μL MeOH was added to a solution of compound 27 ofScheme 2 (5 mg, 8.3 μmmol) in 0.7 mL MeOH at 5° C. The temperature wasallowed to rise to RT gradually. After stirring overnight the mixturewas evaporated and dissolved in 0.7 pyridine. To this solution was addedAc₂O (28 μL, 296 μmmol) at 5° C. The temperature was allowed to rise toRT gradually and after stirring overnight 50 μL H₂O was added. After 3 hthe volatiles was evaporated and the resulting residue was evaporated togive compound 145 (4.7 mg) as a semi solid. MS m/z C₂₂H₄₅N₄O₇S (M+1)⁺calculated 569.3. found 569.

Compound 146

DIEA (6 μL, 34 μmol) and compound 142 (5.5 mg, 8.3 μmol) were added to0.5 mL DMF solution of compound 530 (4.7 mg, 8.3 μmmol) and HATU (3.2mg, 8.4 μmol) at 5° C. After 20 min, the resulting mixture was passedthrough a reverse phase column (ACN:(20 mM NH₄(HCO₃) buffer, pH 7),5-100%) to give compound 146 (4.5 mg) as a white solid. MS m/zC₅₃H₇₉N₁₀O₁₃S (M+1)⁺ calculated 1095.5. found 1095.5. Compound 146 isalso depicted hereinabove as structure (VI-r).

Compound 147

DIEA (1.4 uL, 8 μmol) and a drop of saturated NH₄Cl solution were addedinto a 0.5 mL DMF solution of compound 146 (2 mg, 1.8 μmol) and HATU(1.6 mg, 4.6 μmol). After 10 min the mixture was passed through areverse phase column (ACN:(20 mM NH₄(HCO₃) buffer, pH 7), 5-100%) togive compound 147 (0.5 mg) as a semi solid. MS m/z C₅₃H₈₀N₁₁O₁₂S (M+1)⁺calculated 1094.6. found 1094. Compound 147 is also depicted hereinaboveas structure (VI-s).

Example 27 4-Aminotubuphenylalanine Diastereomers

Compound 122 (Example 25 above) was determined to be a 3:1 mixture ofdiastereomers 148a and 148b as follows.

Compound 122 (10 mg, 0.026 mmol) was dissolved in a 2 mL mixture of TFAand DCM (1:1) at RT. After 3 h the solvents were evaporated and theresidue was passed through a reverse phase column (ACN:H₂O; 0-100% with0.1% TFA) to give a 3:1 mixture of compounds 149a and 149b. The majorisomer in this mixture was assigned structure 149a by comparing the NMRspectrum of the mixture with the NMR of an authentic sample of compound149a made from compound 150.

Concentrated HNO₃ (10 μL) was added to a 200 μL concentrated H₂SO₄solution of tubuphenylalanine 150 (Peltier et al. 2006; 6 mg, 0.025mmol) at 5° C. After 20 min the solution was poured onto 2 mL of cooled7% K₂CO₃ solution. 10 mL EtOAc was then added. After separation, theorganic phase was dried by evaporation and the residue was passedthrough a reverse phase column (ACN:H₂O; 0-100% with 0.1% TFA) to givenitro compound 151 (5 mg). ¹H NMR (400 MHz, CD₃OD) δ 8.27-8.21 (m, 2H),7.56-7.49 (m, 2H), 3.69-3.58 (m, 1H), 3.07 (d, J=7.2 Hz, 2H), 2.73-2.61(m, 1H), 2.04-1.91 (m, 1H), 1.64 (ddd, J=14.7, 8.2, 4.9 Hz, 1H), 1.20(d, J=7.1 Hz, 3H). MS m/z C₁₂H₁₇N₂O₄ (M+1)⁺ calculated 253.1. found 253.

Nitro compound 151 then converted to compound 149a as follows: Nitrocompound 151 (5 mg, 0.01 mmol) was mixed with Pd/C (10 mg, 10%) in 5 mLMeOH at RT. The flask was filled with H₂ using a balloon. After 1 h themixture was filtered and evaporated to give compound 149a (4.5 mg). ¹HNMR (400 MHz, CD₃OD) δ 7.08-7.03 (m, 2H), 6.83-6.79 (m, 2H), 3.51-3.41(m, 1H), 2.84-2.78 (m, 2H), 2.68-2.58 (m, 1H), 2.04-1.92 (m, 1H), 1.60(d, J=7.8 Hz, 1H), 1.18 (d, J=7.0, Hz, 3H). MS m/z C₁₂H₁₉N₂O₂ (M+1)⁺calculated 223.1. found 223. This NMR spectrum was the basis forassigning the structures of the major component of the 148a/148b and149a/149b mixtures.

A sample of compound 122 was passed through a reverse phase column(ACN:H₂O; 0-100% with 0.1% TFA) and the fractions with the minor isomerwere collected and lyophilized. The resulting product was then treatedwith TFA and DCM to remove the Boc group. After 1 h the solvents wereevaporated to give a product, which was assigned to be compound 149b,after comparing its NMR spectrum with that of compound 149a. Compound149b: ¹H NMR (400 MHz, CD₃OD) 7.36-7.23 (m, 2H), 7.22-7.09 (m, 2H),3.59-3.40 (m, 1H), 3.04-2.84 (m, 2H), 2.61-2.45 (m, 1H) 2.07-1.87 (m,1H), 1.73-1.58 (m, 1H), 1.21-1.09 (d, J=7.2 Hz, 3H).

Compounds 148a, 148b, 149a, and 149b can be used to prepare compounds ofthis invention having a 4-aminotubuphenylalanine subunit, where thestereochemistry of the alpha-methyl group is defined, utilizing thesynthetic approaches exemplified above, mutatis mutandis. Compounds 82aand 82b (Example 17) can also be put to similar use.

The foregoing detailed description of the invention includes passagesthat are chiefly or exclusively concerned with particular parts oraspects of the invention. It is to be understood that this is forclarity and convenience, that a particular feature may be relevant inmore than just the passage in which it is disclosed, and that thedisclosure herein includes all the appropriate combinations ofinformation found in the different passages. Similarly, although thevarious figures and descriptions herein relate to specific embodimentsof the invention, it is to be understood that where a specific featureis disclosed in the context of a particular figure or embodiment, suchfeature can also be used, to the extent appropriate, in the context ofanother figure or embodiment, in combination with another feature, or inthe invention in general.

Further, while the present invention has been particularly described interms of certain preferred embodiments, the invention is not limited tosuch preferred embodiments. Rather, the scope of the invention isdefined by the appended claims.

REFERENCES

Full citations for the following references cited in abbreviated fashionby first author (or inventor) and date earlier in this specification areprovided below. Each of these references is incorporated herein byreference for all purposes.

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What is claimed is:
 1. A conjugate having a structure according to theformula

where Ab represents an antibody.
 2. A conjugate according to claim 1,wherein Ab is a monoclonal antibody.
 3. A conjugate according to claim1, wherein Ab is a human monoclonal antibody.
 4. A conjugate accordingto claim 1, wherein Ab is an anti-PSMA (prostate specific membraneantigen) antibody, an anti-CD70 antibody, or an anti-mesothelinantibody.
 5. A conjugate according to claim 1, wherein Ab is ananti-CD70 antibody.
 6. A conjugate according to claim 5, wherein theanti-CD70 antibody is antibody 2H5.
 7. A conjugate according to claim 1,wherein Ab is an anti-mesothelin antibody.
 8. A conjugate according toclaim 7, wherein the anti-mesothelin antibody is antibody 6A4.